C-reactive protein-complement factor H axis as a biomarker of activity in early and intermediate age-related macular degeneration.

Purpose: To determine and compare the serum levels of complement Factor H (FH), monomeric C-Reactive Protein (mCRP) and pentameric C-Reactive protein (pCRP) in patients with age-related macular degeneration (AMD) and to correlate them with clinical, structural and functional parameters. Methods: Cross-sectional observational study. One hundred thirty-nine individuals (88 patients and 51 healthy controls) from two referral centers were included and classified into three groups: early or intermediate AMD (n=33), advanced AMD (n=55), and age and sex matched healthy controls (n=51). Serum levels of FH, mCRP, and pCRP were determined and correlated with clinical and imaging parameters. Results: Patients with intermediate AMD presented FH levels significantly lower than controls [186.5 (72.1-931.8) µg/mL vs 415.2 (106.1-1962.2) µg/mL; p=0.039] and FH levels <200 µg/mL were associated with the presence of drusen and pigmentary changes in the fundoscopy (p=0.002). While no differences were observed in pCRP and mCRP levels, and mCRP was only detected in less than 15% of the included participants, women had a significantly higher detection rate of mCRP than men (21.0% vs. 3.8%, p=0.045). In addition, the ratio mCRP/FH (log) was significantly lower in the control group compared to intermediate AMD (p=0.031). Visual acuity (p<0.001), macular volume (p<0.001), and foveal thickness (p=0.034) were significantly lower in the advanced AMD group, and choroidal thickness was significantly lower in advanced AMD compared to early/intermediate AMD (p=0.023). Conclusion: Intermediate AMD was associated in our cohort with decreased serum FH levels together with increased serum mCRP/FH ratio. All these objective serum biomarkers may suggest an underlying systemic inflammatory process in early/intermediate AMD patients.

Heterozygous mutations in factor H aggravate pathological damage in a stable IgA deposition model induced by Lactobacillus casei cell wall extract.

Introduction: Activation of complement through the alternative pathway (AP) has a key role in the pathogenesis of IgA nephropathy (IgAN). We previously showed, by intraperitoneal injection of Lactobacillus casei cell wall extract (LCWE), C57BL/6 mice develop mild kidney damage in association with glomerular IgA deposition. To further address complement activity in causing glomerular histological alterations as suggested in the pathogenesis of IgAN, here we used mice with factor H mutation (FHW/R) to render AP overactivation in conjunction with LCWE injection to stimulate intestinal production of IgA. Methods: Dose response to LCWE were examined between two groups of FHW/R mice. Wild type (FHW/W) mice stimulated with LCWE were used as model control. Results: The FHW/R mice primed with high dose LCWE showed elevated IgA and IgA-IgG complex levels in serum. In addition to 100% positive rate of IgA and C3, they display elevated biomarkers of kidney dysfunction, coincided with severe pathological lesions, resembling those of IgAN. As compared to wild type controls stimulated by the same high dose LCWE, these FHW/R mice exhibited stronger complement activation in the kidney and in circulation. Discussion: The new mouse model shares many disease features with IgAN. The severity of glomerular lesions and the decline of kidney functions are further aggravated through complement overactivation. The model may be a useful tool for preclinical evaluation of treatment response to complement-inhibitors.

Case report: A family of atypical hemolytic uremic syndrome involving a CFH::CFHR1 fusion gene and CFHR3-1-4-2 gene duplication.

Mutations in the complement factor H (CFH) gene are associated with complement dysregulation and the development of atypical hemolytic uremic syndrome (aHUS). Several fusion genes that result from genomic structural variation in the CFH and complement factor H-related (CFHR) gene regions have been identified in aHUS. However, one allele has both CFHR gene duplication and CFH::CFHR1 fusion gene have not been reported. An 8-month-old girl (proband) presented with aHUS and was treated with ravulizumab. Her paternal grandfather developed aHUS previously and her paternal great grandmother presented with anti-neutrophil cytoplasmic antibody-associated vasculitis and thrombotic microangiopathy (TMA). However, the proband's parents have no history of TMA. A genetic analysis revealed the presence of CFH::CFHR1 fusion gene and a CFHR3-1-4-2 gene duplication in the patient, her father, and her paternal grandfather. Although several fusion genes resulting from structural variations of the CFH-CFHR genes region have been identified, this is the first report of the combination of a CFH::CFHR1 fusion gene with CFHR gene duplication. Because the CFH-CFHR region is highly homologous, we hypothesized that CFHR gene duplication occurred. These findings indicate a novel pathogenic genomic structural variation associated with the development of aHUS.

Protective role of complement factor H against the development of preeclampsia.

Pregnancy is an immunologically regulated, complex process. A tightly controlled complement system plays a crucial role in the successful establishment of pregnancy and parturition. Complement inhibitors at the feto-maternal interface are likely to prevent inappropriate complement activation to protect the fetus. In the present study, we aimed to understand the role of Factor H (FH), a negative regulator of complement activation, in normal pregnancy and in a model of pathological pregnancy, i.e. preeclampsia (PE). The distribution and expression of FH was investigated in placental tissues, various placental cells, and in the sera of healthy (CTRL) or PE pregnant women via immunohistochemistry, RT-qPCR, ELISA, and Western blot. Our results showed a differential expression of FH among the placental cell types, decidual stromal cells (DSCs), decidual endothelial cells (DECs), and extravillous trophoblasts (EVTs). Interestingly, FH was found to be considerably less expressed in the placental tissues of PE patients compared to normal placental tissue both at mRNA and protein levels. Similar results were obtained by measuring circulating FH levels in the sera of third trimester CTRL and PE mothers. Syncytiotrophoblast microvesicles, isolated from the placental tissues of PE and CTRL women, downregulated FH expression by DECs. The present study appears to suggest that FH is ubiquitously present in the normal placenta and plays a homeostatic role during pregnancy.

Factor H-related protein 1 promotes complement-mediated opsonization of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important human opportunistic pathogen responsible for a wide range of infections. The complement system is the main early host defense mechanism to control these infections. P. aeruginosa counteracts complement attack by binding Factor H (FH), a complement regulator that inactivates C3b, preventing the formation of the C3-convertase and complement amplification on the bacterial surface. Factor H-related proteins (FHRs) are a group of plasma proteins evolutionarily related to FH that have been postulated to interfere in this bacterial mechanism of resisting complement. Here, we show that FHR-1 binds to P. aeruginosa via the outer membrane protein OprG in a lipopolysaccharide (LPS) O antigen-dependent manner. Binding assays with purified components or with FHR-1-deficient serum supplemented with FHR-1 show that FHR-1 competes with FH for binding to P. aeruginosa. Blockage of FH binding to C3b deposited on the bacteria reduces FH-mediated cofactor activity of C3b degradation, increasing the opsonization of the bacteria and the formation of the potent chemoattractant C5a. Overall, our findings indicate that FHR-1 is a host factor that promotes complement activation, facilitating clearance of P. aeruginosa by opsonophagocytosis.

Gliosis-dependent expression of complement factor H truncated variants attenuates retinal neurodegeneration following ischemic injury.

Inherited, age-related, and acute retinal diseases are often exacerbated by an aberrant or excessive activity of the complement system. Consequently, cells not directly affected by an acute event or genetic variants may degenerate, resulting in enhanced visual impairment. The therapeutic potential of supplementation of complement factor H (FH), a key regulator of the complement cascade, is therefore particularly promising in the context of retinal diseases caused by complement activation. In this study, we engineered adeno-associated viruses (AAVs) containing sequences of two truncated human FH variants. The expression of these variants was regulated by the glial fibrillary acidic protein (GFAP) promoter, which is selectively active in gliotic Müller cells. Both FH variants consisted of FH domains 19-20, which were connected to domains 1-4 and 1-7, respectively, by a polyglycine linker. These AAVs were intravitreally injected following ischemic injury of C57BL/6J mouse retinas. We observed transgene expression in gliotic Müller cells and to some extent in astrocytes. The expression correlated directly with damage severity. Interventions resulted in decreased complement activation, accelerated normalization of microglia activity and morphological improvements. Reduced levels of C3 transcripts and C3d protein in conjunction with higher transcript levels of inhibitory regulators like Cfi and Cfh, hinted at attenuated complement activity. This study demonstrates the great potential of complement regulatory gene addition therapy. With further in vivo testing it could be applied to treat a wide range of retinal diseases where no causative therapies are available.

Efficacy of GalNAc C3 siRNAs in factor H-deficient mice with C3 glomerulopathy.

Complement alternative pathway (AP) dysregulation drives C3 glomerulopathy (C3G), a rare renal disorder characterized by glomerular C3 deposition and glomerular damage, for which no effective treatments are available. Blockade of complement C3 is emerging as a viable therapeutic option. In an earlier study we showed that SLN500, a small interfering RNA targeting liver C3 synthesis, was able to limit AP dysregulation and glomerular C3d deposits in mice with partial factor H (FH) deficiency (Cfh+/- mice). Here, we assessed the pharmacological effects of SLN501 - an optimized SLN500 version - in mice with complete FH deficiency (Cfh-/- mice) that exhibit a more severe C3G phenotype. SLN501 effectively prevented liver C3 synthesis, thus limiting AP dysregulation, glomerular C3d deposits and the development of ultrastructural alterations. These data provide firm evidence of the use of siRNA-mediated liver C3 gene silencing as a potential therapy for treating C3G patients with either partial or complete FH loss of function.

Mechanisms by which Factor H protects Trypanosoma cruzi from the alternative pathway of complement.

Chagas disease, a chronic disabling disease caused by the protozoan Trypanosoma cruzi, has no standardized treatment or preventative vaccine. The infective trypomastigote form of T. cruzi is highly resistant to killing by the complement immune system. Factor H (FH), a negative regulator of the alternative pathway (AP) of complement on cell surfaces and in blood, contains 20 short consensus repeat domains. The four N-terminal domains of FH inactivate the AP, while the other domains interact with C3b/d and glycan markers on cell surfaces. Various pathogens bind FH to inactivate the AP. T. cruzi uses its trans-sialidase enzyme to transfer host sialic acids to its own surface, which could be one of the approaches it uses to bind FH. Previous studies have shown that FH binds to complement-opsonized T. cruzi and parasite desialylation increases complement-mediated lysis of trypomastigotes. However, the molecular basis of FH binding to T. cruzi remain unknown. Only trypomastigotes, but not epimastigotes (non-infective, complement susceptible) bound FH directly, independent of C3 deposition, in a dose-dependent manner. Domain mapping experiments using 3-5 FH domain fragments showed that domains 5-8 competitively inhibited FH binding to the trypomastigotes by ~35% but did not decrease survival in complement. FH-Fc or mutant FH-Fc fusion proteins (3-11 contiguous FH domains fused to the IgG Fc) also did not kill trypomastigotes. FH-related protein-5, whose domains bear significant sequence identity to all known polyanion-binding FH domains (6-7, 10-14, 19-20), fully inhibited FH binding to trypomastigotes and reduced trypomastigote survival to < 24% in the presence of serum. In conclusion, we have elucidated the role of FH in complement resistance of trypomastigotes.

The human factor H protein family - an update.

Complement is an ancient and complex network of the immune system and, as such, it plays vital physiological roles, but it is also involved in numerous pathological processes. The proper regulation of the complement system is important to allow its sufficient and targeted activity without deleterious side-effects. Factor H is a major complement regulator, and together with its splice variant factor H-like protein 1 and the five human factor H-related (FHR) proteins, they have been linked to various diseases. The role of factor H in inhibiting complement activation is well studied, but the function of the FHRs is less characterized. Current evidence supports the main role of the FHRs as enhancers of complement activation and opsonization, i.e., counter-balancing the inhibitory effect of factor H. FHRs emerge as soluble pattern recognition molecules and positive regulators of the complement system. In addition, factor H and some of the FHR proteins were shown to modulate the activity of immune cells, a non-canonical function outside the complement cascade. Recent efforts have intensified to study factor H and the FHRs and develop new tools for the distinction, quantification and functional characterization of members of this protein family. Here, we provide an update and overview on the versatile roles of factor H family proteins, what we know about their biological functions in healthy conditions and in diseases.

Factor H's Control of Complement Activation Emerges as a Significant and Promising Therapeutic Target for Alzheimer's Disease Treatment.

The complement is a component of the innate immune system designed to fight infections and tissue- or age-related damages. Complement activation creates an inflammatory microenvironment, which enhances cell death. Excessive complement inflammatory activity has been linked to alterations in the structure and functions of the blood-brain barrier, contributing to a poor prognosis for Alzheimer's disease (AD). In the AD preclinical phase, individuals are often clinically asymptomatic despite evidence of AD neuropathology coupled with heightened inflammation. Considering the involvement of the complement system in the risk of developing AD, we hypothesize that inhibiting complement activation could reduce this inflammatory period observed even before clinical signs, thereby slowing down the onset/progression of AD. To validate our hypothesis, we injected complement inhibitor factor H into the brain of APP/PS1 AD mice at early or late stages of this pathology. Our results showed that the injection of factor H had effects on both the onset and progression of AD by reducing proinflammatory IL6, TNF-α, IL1ß, MAC and amyloid beta levels. This reduction was associated with an increase in VGLUT1 and Psd95 synaptic transmission in the hippocampal region, leading to an improvement in cognitive functions. This study invites a reconsideration of factor H's therapeutic potential for AD treatment.

Thrombospondin-1, BIM and CFH polymorphisms and response to anti-VEGF treatment in neovascular age- related macular degeneration patients.

Age-related macular degeneration (AMD) is a vision threatening disease in older adults. Anti-VEGF treatment is effective for the majority of neovascular AMD (nAMD) patients, although approximately 30% of nAMD patients have an incomplete response for unknown reasons. Here we assessed the contribution of single nucleotide polymorphisms (SNPs) in key angioinflammatory regulatory genes in nAMD patients with an incomplete response compared to those responsive to anti-VEGF treatment. A total of 25 responsive and 30 nAMD patients with an incomplete response to anti-vascular endothelial growth factor (anti-VEGF) treatment were examined for known SNPs that impact the structure and function of thromobospondin-1 (TSP1), Bcl-2-interacting mediator of cell death (BIM) and complement factor H (CFH). Plasma levels of C-C motif chemokine ligand 2 (CCL2/MCP1), TSP1 and VEGF were assessed by ELISA. Patients responsive to anti-VEGF treatment showed a significant increase in the TSP1 rs2228262 AA allele and a trend for the BIM (rs724710) CT allele. Consistent with previous reports, 42% of the patients responsive to anti-VEGF expressed the CC allele for CFH rs1061170. Although the CFH TT allele had similarly low prevalence in both groups, the TC allele tended to be more prevalent in patients with an incomplete response. Patients with an incomplete response also had increased plasma CCL2/MCP1 levels, consistent with the role increased inflammation has in the pathogenesis of nAMD. Our studies point to new tools to assess the potential responsiveness of nAMD patients to anti-VEGF treatment and suggest the potential use of anti-CCL2 for treatment of nAMD patients with an incomplete response to anti-VEGF.

The complement factor H-related protein-5 (CFHR5) exacerbates pathological bone formation in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic inflammatory disease, characterized by excessive new bone formation. We previously reported that the complement factor H-related protein-5 (CFHR5), a member of the human factor H protein family, is significantly elevated in patients with AS compared to other rheumatic diseases. However, the pathophysiological mechanism underlying new bone formation by CFHR5 is not fully understood. In this study, we revealed that CFHR5 and proinflammatory cytokines (TNF, IL-6, IL-17A, and IL-23) were elevated in the AS group compared to the HC group. Correlation analysis revealed that CFHR5 levels were not significantly associated with proinflammatory cytokines, while CFHR5 levels in AS were only positively correlated with the high CRP group. Notably, treatment with soluble CFHR5 has no effect on clinical arthritis scores and thickness at hind paw in curdlan-injected SKG, but significantly increased the ectopic bone formation at the calcaneus and tibia bones of the ankle as revealed by micro-CT image and quantification. Basal CFHR5 expression was upregulated in AS-osteoprogenitors compared to control cells. Also, treatment with CFHR5 remarkedly induced bone mineralization status of AS-osteoprogenitors during osteogenic differentiation accompanied by MMP13 expression. We provide the first evidence demonstrating that CFHR5 can exacerbate the pathological bone formation of AS. Therapeutic modulation of CFHR5 could be promising for future treatment of AS. KEY MESSAGES: Serum level of CFHR5 is elevated and positively correlated with high CRP group of AS patients. Recombinant CFHR5 protein contributes to pathological bone formation in in vivo model of AS. CFHR5 is highly expressed in AS-osteoprogenitors compared to disease control. Recombinant CFHR5 protein increased bone mineralization accompanied by MMP13 in vitro model of AS.

C3d-Targeted factor H inhibits tissue complement in disease models and reduces glomerular injury without affecting circulating complement.

Complement-mediated diseases can be treated using systemic inhibitors. However, complement components are abundant in circulation, affecting systemic inhibitors' exposure and efficacy. Furthermore, because of complement's essential role in immunity, systemic treatments raise infection risk in patients. To address these challenges, we developed antibody fusion proteins combining the alternative-pathway complement inhibitor factor H (fH1-5) with an anti-C3d monoclonal antibody (C3d-mAb-2fH). Because C3d is deposited at sites of complement activity, this molecule localizes to tissue complement while minimizing circulating complement engagement. These fusion proteins bind to deposited complement in diseased human skin sections and localize to activated complement in a primate skin injury model. We further explored the pharmacology of C3d-mAb-2fH proteins in rodent models with robust tissue complement activation. Doses of C3d-mAb-2fH >1 mg/kg achieved >75% tissue complement inhibition in mouse and rat injury models while avoiding circulating complement blockade. Glomerular-specific complement inhibition reduced proteinuria and preserved podocyte foot-process architecture in rat membranous nephropathy, indicating disease-modifying efficacy. These data indicate that targeting local tissue complement results in durable and efficacious complement blockade in skin and kidney while avoiding systemic inhibition, suggesting broad applicability of this approach in treating a range of complement-mediated diseases.

Epitope Mapping of Human Polyclonal Antibodies to the fHbp Antigen of a Neisseria Meningitidis Vaccine by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS).

Antigen-antibody interactions play a key role in the immune response post vaccination and the mechanism of action of antibody-based biopharmaceuticals. 4CMenB is a multicomponent vaccine against Neisseria meningitidis serogroup B in which factor H binding protein (fHbp) is one of the key antigens. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to identify epitopes in fHbp recognized by polyclonal antibodies (pAb) from two human donors (HDs) vaccinated with 4CMenB. Our HDX-MS data reveal several epitopes recognized by the complex mixture of human pAb. Furthermore, we show that the pAb from the two HDs recognize the same epitope regions. Epitope mapping of total pAb and purified fHbp-specific pAb from the same HD reveals that the two antibody samples recognize the same main epitopes, showing that HDX-MS based epitope mapping can, in this case at least, be performed directly using total IgG pAb samples that have not undergone Ab-selective purification. Two monoclonal antibodies (mAb) were previously produced from B-cell repertoire sequences from one of the HDs and used for epitope mapping of fHbp with HDX-MS. The epitopes identified for the pAb from the same HD in this study, overlap with the epitopes recognized by the two individual mAbs. Overall, HDX-MS epitope mapping appears highly suitable for simultaneous identification of epitopes recognized by pAb from human donors and to thus both guide vaccine development and study basic human immunity to pathogens, including viruses.

Borreliella burgdorferi factor H-binding proteins are not required for serum resistance and infection in mammals.

The causative agent of Lyme disease (LD), Borreliella burgdorferi, binds factor H (FH) and other complement regulatory proteins to its surface. B. burgdorferi B31 (type strain) encodes five FH-binding proteins (FHBPs): CspZ, CspA, and the OspE paralogs OspEBBN38, OspEBBL39, and OspEBBP38. This study assessed potential correlations between the production of individual FHBPs, FH-binding ability, and serum resistance using a panel of infectious B. burgdorferi clonal populations recovered from dogs. FHBP production was assessed in cultivated spirochetes and by antibody responses in naturally infected humans, dogs, and eastern coyotes (wild canids). FH binding specificity and sensitivity to dog and human serum were also assessed and compared. No correlation was observed between the production of individual FHBPs and FH binding with serum resistance, and CspA was determined to not be produced in animals. Notably, one or more clones isolated from dogs lacked CspZ or the OspE proteins (a finding confirmed by genome sequence determination) and did not bind FH derived from canines. The data presented do not support a correlation between FH binding and the production of individual FHBPs with serum resistance and infectivity. In addition, the limited number and polymorphic nature of cp32s in B. burgdorferi clone DRI85A that were identified through genome sequencing suggest no strict requirement for a defined set of these replicons for infectivity. This study reveals that the immune evasion mechanisms employed by B. burgdorferi are diverse, complex, and yet to be fully defined.

Levels of complement factor H-related 4 protein do not influence susceptibility to age-related macular degeneration or its course of progression.

Dysregulation of the alternative pathway (AP) of the complement system is a significant contributor to age-related macular degeneration (AMD), a primary cause of irreversible vision loss worldwide. Here, we assess the contribution of the liver-produced complement factor H-related 4 protein (FHR-4) to AMD initiation and course of progression. We show that FHR-4 variation in plasma and at the primary location of AMD-associated pathology, the retinal pigment epithelium/Bruch's membrane/choroid interface, is entirely explained by three independent quantitative trait loci (QTL). Using two distinct cohorts composed of a combined 14,965 controls and 20,741 cases, we ascertain that independent QTLs for FHR-4 are distinct from variants causally associated with AMD, and that FHR-4 variation is not independently associated with disease. Additionally, FHR-4 does not appear to influence AMD progression course among patients with disease driven predominantly by AP dysregulation. Modulation of FHR-4 is therefore unlikely to be an effective therapeutic strategy for AMD.

Anti-factor B antibodies in atypical hemolytic uremic syndrome.

BACKGROUND: The etiology of atypical hemolytic uremic syndrome (aHUS) is unknown in 30-40% of patients. Anti-factor B (FB) antibodies are reported in C3 glomerulopathy (C3G) and immune-complex membranoproliferative glomerulonephritis (IC-MPGN), though not in aHUS. METHODS: We screened patients < 18-year-old from cohorts of aHUS and C3G/idiopathic IC-MPGN. Anti-FB IgG antibodies were measured by ELISA and confirmed by Western blot. Normative levels were based on antibody levels in 103 healthy blood donors. RESULTS: Prevalence of anti-FB antibodies was 9.7% (95% CI 6.1-14.5%; n = 21) in 216 patients with aHUS, including 11.5% (95% CI 6.4-18.5%; n = 14) in anti-FH associated aHUS and 11.8% (95% CI 4.4-23.9%; n = 6) in patients without a definitive genetic or autoimmune etiology. Patients with significant genetic variants did not show anti-FB antibodies. In patients with concomitant anti-FB and anti-FH antibodies, median anti-FH titers were higher (11,312 AU/mL vs. 4920 AU/mL; P = 0.04). Anti-FB antibody titer correlated with disease severity (hemoglobin and platelets; P < 0.05), declined following plasma exchange and increased during relapse. While 4/64 patients with C3G (6.3%) and 1/17 with IC-MPGN showed anti-FB antibodies, titers were higher in aHUS (544.8 AU/mL vs. 1028.8 AU/mL; P = 0.003). CONCLUSION: Anti-FB antibodies are present in 6-10% of patients with aHUS and C3G/IC-MPGN, with higher titers in the former. The diagnostic and therapeutic implication of anti-FB antibodies in aHUS needs confirmation and further studies. The study shows propensity for autoantibody generation and co-existence of multiple risk factors for aHUS in Indian children.

Complement dysregulation associated with a genetic variant in factor H-related protein 5 in atypical hemolytic uremic syndrome.

BACKGROUND: Atypical hemolytic uremic syndrome (aHUS) can be associated with mutations, deletions, or hybrid genes in factor H-related (FHR) proteins. METHODS: A child with aHUS was investigated. Genetics was assessed by Sanger and next generation sequencing. Serum FHR5 was evaluated by immunoblotting, ELISA, and by induction of rabbit red blood cell hemolysis in the presence/absence of recombinant human rFHR5. Mutagenesis was performed in HEK cells. RESULTS: A heterozygous genetic variant in factor H-related protein 5 (CFHR5), M514R, was found in the child, who also had a homozygous deletion of CFHR3/CFHR1, and antibodies to factor H, as well as low levels of C3. Patient serum exhibited low levels of FHR5. In the presence of rabbit red blood cells, patient serum induced hemolysis which decreased when rFHR5 was added at physiological concentrations. Similar results were obtained using serum from the father, bearing the CFHR5 variant without factor H antibodies. Patient FHR5 formed normal dimers. The CFHR5 M514R variant was expressed in HEK cells and minimal secretion was detected whereas the protein level was elevated in cell lysates. CONCLUSIONS: Decreased secretion of the product of the mutant allele could explain the low FHR5 levels in patient serum. Reduced hemolysis when rFHR5 was added to serum suggests a regulatory role regarding complement activation on red blood cells. As such, low levels of FHR5, as demonstrated in the patient, may contribute to complement activation.

Whole genome sequencing of 4,787 individuals identifies gene-based rare variants in age-related macular degeneration.

Genome-wide association studies have contributed extensively to the discovery of disease-associated common variants. However, the genetic contribution to complex traits is still largely difficult to interpret. We report a genome-wide association study of 2394 cases and 2393 controls for age-related macular degeneration (AMD) via whole-genome sequencing, with 46.9 million genetic variants. Our study reveals significant single-variant association signals at four loci and independent gene-based signals in CFH, C2, C3, and NRTN. Using data from the Exome Aggregation Consortium (ExAC) for a gene-based test, we demonstrate an enrichment of predicted rare loss-of-function variants in CFH, CFI, and an as-yet unreported gene in AMD, ORMDL2. Our method of using a large variant list without individual-level genotypes as an external reference provides a flexible and convenient approach to leverage the publicly available variant datasets to augment the search for rare variant associations, which can explain additional disease risk in AMD.