C3 Variant Research Articles

A Cell-Based Assay to Measure the Activity of the Complement Convertases

IntroductionThe complement system serves as a crucial defense mechanism against invading pathogens, but dysregulation of this system can result in harmful consequences. Central to the complement cascade are the classical/lectin and the alternative pathway convertases. Aberrant regulation of the convertases is often implicated in the development of rare complement-related diseases. However, analyzing convertase activity poses a significant challenge due to their labile nature and intricate interactions with serum proteins. MethodsIn this study, we propose a novel assay for the functional evaluation of these complexes. Our approach leverages a widely available human lymphoma cell line, which, when sensitized with antibodies, triggers activation of the classical pathway with a substantial amplification by the alternative pathway. The combined action of two C5 blockers eculizumab and crovalimab let the cascade proceed up to the level of convertases but not further. In the next step, C5 inhibitors are washed away and guinea pig serum in EDTA buffer supports the development of lytic sites on the platform of pre-existing convertases. ResultsThe assay detects recombinant gain-of-function components of both convertase types within human serum or plasma. Furthermore, we demonstrate the assay's practical utility in analyzing nephrological patients harboring C3 genetic variants and illustrate its capacity to distinguish between patients and asymptomatic relatives carrying the same pathogenic C3 variant. ConclusionsWe provided a proof of concept of a new assay that detects convertase overactivity in individuals carrying variants of both pathogenic character or these of unknown significance in ubiquitous complement proteins like C3.

A combinatorial strategy for HRV 3C protease engineering to achieve the N-terminal free cleavage

Human rhinovirus 3C protease (HRV 3CP) has a high specificity against the substrate of LEVLFQ↓G at P1′ site, which plays an important role in biotechnology and academia as a fusion tag removal tool. However, a non-ignorable limitation is that an extra residue of Gly would remain at the N terminus of the recombinant target protein after cleavage with HRV 3CP, thus potentially causing protein mis-functionality or immunogenicity. Here, we developed a combinatorial strategy by integrating structure-guided library design and high-throughput screening of eYESS approach for HRV 3CP engineering to expand its P1′ specificity. Finally, a C3 variant was obtained, exhibiting a broad substrate P1′ specificity to recognize 20 different amino acids with the highest activity against LEVLFQ↓M (kcat/KM = 3.72 ± 0.04 mM−1∙s−1). Further biochemical and NGS-mediated substrate profiling analysis showed that C3 variant still kept its substrate stringency at P1 site and good residue tolerance at P2′ site, but with an expanded P1′ specificity. Structural simulation of C3 indicated a reconstructed S1′ binding pocket as well as new interactions with the substrates. Overall, our studies here prompt not only the practical applications and understanding of substrate recognition mechanisms of HRV 3CP, also provide new tools for other enzyme engineering.

Characterization of Compost Made from hen Manure and Wood Ash and Its Effect on Soil Properties, Production and Nutritional Quality of Tomato (Lycopersicon esculentum Mill.)

The effect of a compost based on chicken manure and wood ash on the production and nutritional quality of tomatoes was evaluated. The mixtures of the constituents of each compost were made at variable m/m proportions: 25/75 (C1); 50/50 (C2); 75/25 (C3) and 100/0 (C4). Tomato cultivation in pots was done in completely randomized random blocks with 5 repetitions, and 3 compost/soil m/m proportions (5%, 10% and 15%). The physicochemical and biochemical parameters of the compost and the amended soil, as well as the production and nutritional quality of the fruits, were evaluated. The compost showed higher pH values than the unamended soil. The C3 variant exhibited the highest pH (9.97±0.00). The C/N ratio varied from 14.78±0.67 to 15.30±0.35 values higher than that of the unamended soil (11.86±0.27); exchangeable ion concentrations were higher in the compost. The microbial biomass and the enzyme activities of the compost were significantly higher than those of the unamended soil, the greatest values were obtained with the C3 variant. pH, EC, C/N ratio, and exchangeable ion concentrations were higher in all treated soils than in soils amended with chemical fertilizer. Tomato production was higher with the variant (15% C3) with a value of 156.00 ± 1.00 fruits, the soil amended with chemical fertilizer having given 14.00 ± 1.00 fruits. Biological tomatoes obtained with compost were richer in lycopene, vitamin C, phenolic compounds and minerals than those obtained with chemical inputs (Chemical tomatoes). The compost has improved the production and nutritional quality of the tomato, which is an efficient alternative for sustainable agriculture.

Compost Made from Cow Dung and Sawdust Improves the Physicochemical and Biochemical Properties of the Soil, the Production and the Nutritional Quality of Tomatoes (Lycopersicon esculentum Mill.)

The effect of a compost based on cow dung and sawdust on the production and nutritional quality of tomatoes was evaluated. The compost was prepared by mixing the different constituents in variable mass/mass proportions: 25/75, 50/50, 75/25 and 100/0. The experimental device for growing tomatoes in pots was made in completely randomized random blocks with 5 repetitions, and 3 compost/soil mass/mass proportions (5%, 10% and 15%) for each variant. The physicochemical, biological and biochemical parameters of the compost and amended soils, the production and the nutritional quality of the fruits were evaluated. The C3 variant of compost showed the highest pH (9.88 ± 0.01); C4 presented the highest C/N ratio (21.45 ± 0.42); C2 showed the highest contents of nitrogen, phosphorus, magnesium, calcium, potassium, and sodium, the highest values of bacterial and fungal flora and cellulase, protease, β-glucosidase, acid and alkaline phosphatase activities. Analysis of amended soils showed that: C2 (15%) has the highest pH. C1 (10%) presented the highest C/N ratio (42.12 ± 3.98) and C3 (15%) the highest contents of nitrogen, magnesium ions and calcium. C4 (15%) presented the highest potassium and sodium content. The compost significantly improved tomato production. The C2 (15%) compost was the most productive with 154 ± 3.00 fruits, 10 times more than the tomato produced with chemical fertilizer (15.00 ± 1.00 fruits). The Biological tomato showed higher proportions of ash, total sugars, lycopene, vitamin C and polyphenols than the chemical tomato. The results show that the organic treatment improved the physicochemical, biological and biochemical properties of the soil, the production and the nutritional quality of the tomato. The compost constitutes therefore an efficient method for sustainable agriculture.

COVID-19 Associated Atypical Hemolytic Uremic Syndrome

Introduction: Atypical hemolytic uremic syndrome (atypical HUS) is a complement-mediated thrombotic microangiopathy (TMA) that affects multiple organs including the kidneys. We present a case of 37-year-old male with recent COVID-19 infection who presented with epistaxis. His blood work was consistent with TMA and acute kidney injury (AKI). Hemodialysis and plasmapheresis (PLEX) were initiated for presumed thrombotic thrombocytopenic purpura (TTP) but after excluding ADAMTS-13 deficiency, eculizumab was started for possible atypical HUS. Renal biopsy and genetic testing ultimately confirmed diagnosis of atypical HUS. Case presentation: A 37-year-old male with past medical history of recent COVID infection presented to the emergency room with complaint of epistaxis. Physical exam showed mild epistaxis from right naris and bilateral leg swelling. Initial laboratory findings were hemoglobin (Hb) 5 g/dL, platelets 70 B/L, Lactate dehydrogenase (LDH) 581 IU/L, haptoglobin < 10 mg/dL, creatinine (Cr) 36.80 mg/dL, blood urea nitrogen (BUN) 232 mg/dL, potassium (K) 6.6 mmol/L, calcium (Ca) 6 mg/dL and normal prothrombin time (PT) and partial thromboplastin time (PTT). Complement C3 levels were low but normal C4 levels. Peripheral blood smear showed schistocytes confirming TMA. He was started on both PLEX and hemodialysis (HD) along with prednisone 80 mg daily. PLEX was stopped on day 4 after normal ADAMTS-13 levels resulted, helping rule out TTP. Based on history and laboratory findings, diagnosis of atypical HUS was favored and eculizumab was started. He received meningococcal vaccine and prophylactic penicillin while on eculizumab. Hemodialysis was continued with eventual improvement of his labs to Hb 7, platelets 119, LDH 339, haptoglobin < 10, Cr 9.07, BUN 85, K 5.8, and Ca 8.0. He was discharged on bimonthly dose of eculizumab and hemodialysis three times a week. Two months later, his labs were Hb 10.4, platelets 184, LDH 490, haptoglobin < 10, Cr 11.60, BUN 82, K 5.2, and Ca 9.2. PT and PTT remained normal. Given persistence of laboratory findings, renal biopsy and genetic testing for complement proteins was done. Kidney biopsy showed diffuse global renal microangiopathy with negative C3. Genetic testing showed a heterozygous C3 variant, homozygous deletion of CFHR1-CFHR4, negative factor H autoantibodies (FHAA), and single nucleotide variant in C5 gene. Discussion: Atypical HUS is a disease characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia, and AKI. The complement system is a part of the immune system that enhances the ability of phagocytic cells to clear microbes and damaged cells. Atypical HUS is caused by dysregulation of the alternative complement pathway that leads to microvascular thrombosis. Various mutations in complement and its regulatory proteins have been discovered in atypical HUS patients. Heterozygous C3 variants result in persistently low C3 levels and cause severe disease. Most individuals with a homozygous deletion of CFHR1 and/or CFHR3, CFHR4 genes have a pathogenic role in the development of the anti-factor H autoantibodies. These autoantibodies interfere with the binding of CFH to the C3 convertase and are associated with defective CFH-dependent cell protection. Some case reports of COVID-19 patients showed that complement activation was significantly stronger in lung alveolar septa, hyaline membranes, and in alveolar lumina via the lectin and alternative complement pathways. Treatment of atypical HUS is symptomatic but in severe disease eculizumab or ravulizumab have roles by binding to the complement protein C5, inhibiting its cleavage to C5a and C5b, and preventing formation of terminal complement complex C5b-9. Patients on these therapies are at risk of meningococcal disease, thus it is recommended to get meningococcal vaccines and prophylactic penicillin for the duration of therapy. Our patient had homozygous deletion of CFHR1-CFHR4 genes with no factor H autoantibodies. However, he did have C3 variant with gain of function mutation which caused overt activation of alternative pathway as indicated by his low C3 levels with normal C4 levels. He responded partially to eculizumab and discussion on possible kidney transplant has been started.

POS-029 RARE VARIANTS IN COMPLEMENT GENES AND OUTCOMES OF C3 GLOMERULOPATHY AND IMMUNOGLOBULIN-MEDIATED MPGN

IntroductionC3 glomerulopathy (C3G) and idiopathic immunoglobulin-mediated membranoproliferative glomerulonephritis (Ig-MPGN) are rare kidney diseases mediated by dysregulation of the complement alternative pathway. In most cases, complement overactivation is acquired due to the presence of autoantibody targeting C3 convertase. C3G/Ig-MPGN with inherited background are even rarer. However, specific presentation at diagnosis and clinical courses on native kidney and after kidney transplantation are not well known.MethodsRare variant in CFH, CFI and C3 genes (defined by minor allelic frequency bellow 0.1%) were screened in a large national series of 398 patients with C3G (n=296) or Ig-MPGN (n=102) We described genetic, immunological, clinical data at diagnosis and renal outcomes of patients.ResultsView Large Image Figure ViewerDownload Hi-res image Download (PPT)ConclusionsHere, we reported exhaustive clinical, histological and immunological features of the largest series of patients with C3G and Ig-MPGN associated with CFH, CFI or C3 rare variants. Despite an important heterogeneity of clinical features, our result highlights the severity of inherited C3G/Ig-MPGN associated with CFI, CFH or C3 variant, and in particular associated with CFI and C3 variant with early and high degree of recurrence after transplantation.No conflict of interest IntroductionC3 glomerulopathy (C3G) and idiopathic immunoglobulin-mediated membranoproliferative glomerulonephritis (Ig-MPGN) are rare kidney diseases mediated by dysregulation of the complement alternative pathway. In most cases, complement overactivation is acquired due to the presence of autoantibody targeting C3 convertase. C3G/Ig-MPGN with inherited background are even rarer. However, specific presentation at diagnosis and clinical courses on native kidney and after kidney transplantation are not well known. C3 glomerulopathy (C3G) and idiopathic immunoglobulin-mediated membranoproliferative glomerulonephritis (Ig-MPGN) are rare kidney diseases mediated by dysregulation of the complement alternative pathway. In most cases, complement overactivation is acquired due to the presence of autoantibody targeting C3 convertase. C3G/Ig-MPGN with inherited background are even rarer. However, specific presentation at diagnosis and clinical courses on native kidney and after kidney transplantation are not well known. MethodsRare variant in CFH, CFI and C3 genes (defined by minor allelic frequency bellow 0.1%) were screened in a large national series of 398 patients with C3G (n=296) or Ig-MPGN (n=102) We described genetic, immunological, clinical data at diagnosis and renal outcomes of patients. Rare variant in CFH, CFI and C3 genes (defined by minor allelic frequency bellow 0.1%) were screened in a large national series of 398 patients with C3G (n=296) or Ig-MPGN (n=102) We described genetic, immunological, clinical data at diagnosis and renal outcomes of patients. Results ConclusionsHere, we reported exhaustive clinical, histological and immunological features of the largest series of patients with C3G and Ig-MPGN associated with CFH, CFI or C3 rare variants. Despite an important heterogeneity of clinical features, our result highlights the severity of inherited C3G/Ig-MPGN associated with CFI, CFH or C3 variant, and in particular associated with CFI and C3 variant with early and high degree of recurrence after transplantation.No conflict of interest Here, we reported exhaustive clinical, histological and immunological features of the largest series of patients with C3G and Ig-MPGN associated with CFH, CFI or C3 rare variants. Despite an important heterogeneity of clinical features, our result highlights the severity of inherited C3G/Ig-MPGN associated with CFI, CFH or C3 variant, and in particular associated with CFI and C3 variant with early and high degree of recurrence after transplantation.

Atypical hemolytic and uremic syndrome due to C3 mutation in pancreatic islet transplantation: a case report

BackgroundWe here report on the first observation of a C3 mutation that is related to atypical hemolytic and uremic syndrome (aHUS), which occurred in a pancreatic islet transplant patient. Immunosuppressive treatments, such as calcineurin inhibitors, have been linked to undesirable effects like nephrotoxicity.Case presentationA 40-year-old man with brittle diabetes, who was included in the TRIMECO trial, became insulin-independent 2 months after pancreatic islet transplantation. About 15 months after islet transplantation, the patient exhibited acute kidney injury due to aHUS. Despite plasma exchange and eculizumab treatment, the patient developed end-stage renal disease.A genetic workup identified a missense variant (p.R592Q) in the C3 gene. In vitro, this C3 variant had defective Factor I proteolytic activity with membrane proteins as cofactor proteins, which was thus classified as pathogenic. About 1 year after the aHUS episode, kidney transplantation was carried out under the protection of the specific anti-C5 monoclonal antibody eculizumab. The patient had normal kidney function, with preserved pancreatic islet function 4 years later.ConclusionsPancreatic islet transplantation could have triggered this aHUS episode, but this link needs to be clarified. Although prophylactic eculizumab maintains kidney allograft function, its efficacy still needs to be studied in larger populations.

Convergent effects of a functional C3 variant on brain atrophy, demyelination, and cognitive impairment in multiple sclerosis

Background: Complement system activation products are present in areas of neuroinflammation, demyelination, and neurodegeneration in brains of patients with multiple sclerosis (MS). C3 is a central element in the activation of complement cascades. A common coding variant in the C3 gene (rs2230199, C3R102G) affects C3 activity. Objectives: To assess the effects of rs2230199 on MS severity using clinical, cognitive, and imaging measures. Methods: In total, 161 relapse-onset MS patients (Expanded Disability Status Scale (EDSS) ≤ 6) underwent physical assessments, cognitive tests (Paced Auditory Serial Addition Test (PASAT), Symbol Digit Modalities Test (SDMT), and California Verbal Learning Test (CVLT)), and magnetic resonance imaging (MRI). Lesion volumes were quantified semi-automatically. Voxel-wise analyses were performed to assess the effects of rs2230199 genotype on gray matter (GM) atrophy (n = 155), white matter (WM) fractional anisotropy (FA; n = 105), and WM magnetization transfer ratio (MTR; n = 90). Results: While rs2230199 minor-allele dosage (C3-102G) showed no significant effect on EDSS and Multiple Sclerosis Functional Composite (MSFC), it was associated with worse cognitive performance (p = 0.02), lower brain parenchymal fraction (p = 0.003), and higher lesion burden (p = 0.02). Moreover, voxel-wise analyses showed lower GM volume in subcortical structures and insula, and lower FA and MTR in several WM areas with higher copies of rs2230199 minor allele. Conclusion: C3-rs2230199 affects white and GM damage as well as cognitive impairment in MS patients. Our findings support a causal role for complement system activity in the pathophysiology of MS.

Symbiont community stability through severe coral bleaching in a thermally extreme lagoon

Coral reefs are threatened by climate change as coral-algal symbioses are currently living close to their upper thermal limits. The resilience of the algal partner plays a key role in determining the thermal tolerance of the coral holobiont and therefore, understanding the acclimatory limits of present day coral-algal symbioses is fundamental to forecasting corals’ responses to climate change. This study characterised the symbiont community in a highly variable and thermally extreme (Max = 37.5 °C, Min = 16.8 °C) lagoon located in the southern Persian/Arabian Gulf using next generation sequencing of ITS2 amplicons. Despite experiencing extreme temperatures, severe bleaching and many factors that would be expected to promote the presence of, or transition to clade D dominance, the symbiont communities of the lagoon remain dominated by the C3 variant, Symbiodinium thermophilum. The stability of this symbiosis across multiple genera with different means of symbiont transmission highlights the importance of Symbiodinium thermophilum for corals living at the acclimatory limits of modern day corals. Corals in this extreme environment did not undergo adaptive bleaching, suggesting they are living at the edge of their acclimatory potential and that this valuable source of thermally tolerant genotypes may be lost in the near future under climate change.

Host specificity of Symbiodinium variants revealed by an ITS2 metahaplotype approach.

Analysis of the widely used ITS region is confounded by the presence of intragenomic variants (IGVs). In Symbiodinium, the algal symbionts of reef building corals, deep-sequencing analyses are used to characterise communities within corals, yet these analyses largely overlook IGVs. Here we consider that distinct ITS2 sequences could represent IGVs rather than distinct symbiont types and argue that symbionts can be distinguished by their proportional composition of IGVs, described as their ITS2 metahaplotype. Using our metahaplotype approach on Minimum Entropy Decomposition (MED) analysis of ITS2 sequences from the corals Acropora downingi, Cyphastrea microphthalma and Playgyra daedalea, we show the dominance of a single species-specific Symbiodinium C3 variant within each coral species. We confirm the presence of these species-specific symbionts using the psbA non-coding region. Our findings highlight the importance of accounting for IGVs in ITS2 analyses and demonstrate their capacity to resolve biological patterns that would otherwise be overlooked.

A common complement C3 variant is associated with protection against wet age-related macular degeneration in a Japanese population.

BackgroundGenetic variants in the complement component 3 gene (C3) have been shown to be associated with age-related macular degeneration (AMD) in Caucasian populations of European descent. In particular, a nonsynonymous coding variant, rs2230199 (R102G), is presumed to be the most likely causal variant in the C3 locus based on strong statistical evidence for disease association and mechanistic functional evidence. However, the risk allele is absent or rare (<1%) in Japanese and Chinese populations, and the association of R102G with AMD has not been reported in Asian populations. Genetic heterogeneity of disease-associated variants among different ethnicities is common in complex diseases. Here, we sought to examine whether other common variants in C3 are associated with wet AMD, a common advanced-stage manifestation of AMD, in a Japanese population.Methodology/Principal FindingsWe genotyped 13 tag single nucleotide polymorphisms (SNPs) that capture the majority of common variations in the C3 locus and tested for associations between these SNPs and wet AMD in a Japanese population comprising 420 case subjects and 197 controls. A noncoding variant in C3 (rs2241394) exhibited statistically significant evidence of association (allelic P = 8.32×10−4; odds ratio = 0.48 [95% CI = 0.31–0.74] for the rs2241394 C allele). Multilocus logistic regression analysis confirmed that the effect of rs2241394 was independent of the previously described loci at ARMS2 and CFH, and that the model including variants in ARMS2 and CFH plus C3 rs2241394 provided a better fit than the model without rs2241394. We found no evidence of epistasis between variants in C3 and CFH, despite the fact that they are involved in the same biological pathway.ConclusionsOur study provides evidence that C3 is a common AMD-associated locus that transcends racial boundaries and provides an impetus for more detailed genetic characterization of the C3 locus in Asian populations.

Production of Candida antarctica Lipase B Gene Open Reading Frame using Automated PCR Gene Assembly Protocol on Robotic Workcell and Expression in an Ethanologenic Yeast for use as Resin-Bound Biocatalyst in Biodiesel Production

A synthetic Candida antarctica lipase B (CALB) gene open reading frame (ORF) for expression in yeast was constructed, and the lycotoxin-1 (Lyt-1) C3 variant gene ORF, potentially to improve the availability of the active enzyme at the surface of the yeast cell, was added in frame with the CALB ORF using an automated PCR assembly and DNA purification protocol on an integrated robotic workcell. Saccharomyces cerevisiae strains expressing CALB protein or CALB Lyt-1 fusion protein were first grown on 2% (w/v) glucose, producing 9.3 g/L ethanol during fermentation. The carbon source was switched to galactose for GAL1-driven expression, and the CALB and CALB Lyt-1 enzymes expressed were tested for fatty acid ethyl ester (biodiesel) production. The synthetic enzymes catalyzed the formation of fatty acid ethyl esters from ethanol and either corn or soybean oil. It was further demonstrated that a one-step-charging resin, specifically selected for binding to lipase, was capable of covalent attachment of the CALB Lyt-1 enzyme, and that the resin-bound enzyme catalyzed the production of biodiesel. High-level expression of lipase in an ethanologenic yeast strain has the potential to increase the profitability of an integrated biorefinery by combining bioethanol production with coproduction of a low-cost biocatalyst that converts corn oil to biodiesel.

Aberrant Receptor Internalization and Enhanced FRS2-dependent Signaling Contribute to the Transforming Activity of the Fibroblast Growth Factor Receptor 2 IIIb C3 Isoform

Alternative splice variants of fibroblast growth factor receptor 2 (FGFR2) IIIb, designated C1, C2, and C3, possess progressive reduction in their cytoplasmic carboxyl termini (822, 788, and 769 residues, respectively), with preferential expression of the C2 and C3 isoforms in human cancers. We determined that the progressive deletion of carboxyl-terminal sequences correlated with increasing transforming potency. The highly transforming C3 variant lacks five tyrosine residues present in C1, and we determined that the loss of Tyr-770 alone enhanced FGFR2 IIIb C1 transforming activity. Because Tyr-770 may compose a putative YXXL sorting motif, we hypothesized that loss of Tyr-770 in the 770YXXL motif may cause disruption of FGFR2 IIIb C1 internalization and enhance transforming activity. Surprisingly, we found that mutation of Leu-773 but not Tyr-770 impaired receptor internalization and increased receptor stability and activation. Interestingly, concurrent mutations of Tyr-770 and Leu-773 caused 2-fold higher transforming activity than caused by the Y770F or L773A single mutations, suggesting loss of Tyr and Leu residues of the 770YXXL773 motif enhances FGFR2 IIIb transforming activity by distinct mechanisms. We also determined that loss of Tyr-770 caused persistent activation of FRS2 by enhancing FRS2 binding to FGFR2 IIIb. Furthermore, we found that FRS2 binding to FGFR2 IIIb is required for increased FRS2 tyrosine phosphorylation and enhanced transforming activity by Y770F mutation. Our data support a dual mechanism where deletion of the 770YXXL773 motif promotes FGFR2 IIIb C3 transforming activity by causing aberrant receptor recycling and stability and persistent FRS2-dependent signaling.

Complement C3 Variant and the Risk of Age-Related Macular Degeneration

Age-related macular degeneration is the most common cause of blindness in Western populations. Susceptibility is influenced by age and by genetic and environmental factors. Complement activation is implicated in the pathogenesis. We tested for an association between age-related macular degeneration and 13 single-nucleotide polymorphisms (SNPs) spanning the complement genes C3 and C5 in case subjects and control subjects from the southeastern region of England. All subjects were examined by an ophthalmologist and had independent grading of fundus photographs to confirm their disease status. To test for replication of the most significant findings, we genotyped a set of Scottish cases and controls. The common functional polymorphism rs2230199 (Arg80Gly) in the C3 gene, corresponding to the electrophoretic variants C3S (slow) and C3F (fast), was strongly associated with age-related macular degeneration in both the English group (603 cases and 350 controls, P=5.9x10(-5)) and the Scottish group (244 cases and 351 controls, P=5.0x10(-5)). The odds ratio for age-related macular degeneration in C3 S/F heterozygotes as compared with S/S homozygotes was 1.7 (95% confidence interval [CI], 1.3 to 2.1); for F/F homozygotes, the odds ratio was 2.6 (95% CI, 1.6 to 4.1). The estimated population attributable risk for C3F was 22%. Complement C3 is important in the pathogenesis of age-related macular degeneration. This finding further underscores the influence of the complement pathway in the pathogenesis of this disease.

Transforming Potential of Alternatively Spliced Variants of Fibroblast Growth Factor Receptor 2 in Human Mammary Epithelial Cells

A breast cancer cell line developed in our laboratory (SUM-52PE) has a 12-fold amplification and high-level overexpression of the oncogene fibroblast growth factor receptor 2 (FGFR2). Previously, nine different alternatively spliced FGFR2 variants were isolated from this cell line. Overexpression of two variants that differ only in their carboxyl termini (C1 and C3) has been successfully accomplished in the immortalized human mammary epithelial cell line H16N2. FGFR2 expression led to the activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling cascades. Phosphorylation of the adapter protein FGF receptor substrate 2 is much more robust in the cells expressing the C3 variant of FGFR2 compared with the C1 variant. H16N2 cells expressing the full-length FGFR2 with the C1 or C3 carboxyl terminus were tested for their ability to grow under epidermal growth factor (EGF)-independent conditions, in soft agar, and for their ability to invade naturally occurring basement membranes and compared with the parental SUM-52PE cell line. All three cell lines grew under EGF-independent conditions and all were inhibited by the FGFR family specific inhibitor PD173074. The full-length FGFR2-C1 and FGFR2-C3 variants grew robustly in soft agar similar to the parental cell line SUM-52PE. However, cells expressing the C3 variant formed large colonies in agar in both insulin-free and EGF-free medium, whereas the cells expressing the C1 variant required insulin for growth. Soft agar growth was also inhibited by PD173074. Because SUM-52PE was developed from a metastatic breast carcinoma, the FGFR2-overexpressing cell lines were assessed for their ability to invade sea urchin embryo cell membranes. H16N2 cells expressing the C1 carboxyl terminus failed to invade sea urchin embryo cell membranes. By contrast, FGFR2-C3-expressing cells were as invasive as the SUM-52 breast cancer cells and erbB-2-overexpressing H16N2 cells. These results indicate that FGFR2 is a transforming oncogene in human mammary epithelial cells when expressed to levels similar to that found in breast cancer cells with FGFR2 gene amplification. Furthermore, the results suggest that different splice variants have differing transforming activities and that signaling from variants expressing the C3 carboxyl terminus results in more autonomous signaling, cell growth, and invasion.

DNA polymorphisms and haplotypes in the human transferrin gene.

Although a large number of human serum transferrin (TF) variants have been described, only one RFLP (AvaI) has so far been found. Here we report three new RFLPs (MvaI in intron 5 and exon 7, BbvI in exon 7) and correlations between RFLPs and between RFLPs and serum TF types. There were strong, but not always complete, disequilibria between RFLP and serum protein alleles. Thus, the most common serum TF variant, C1, was heterogeneous and could be subdivided into two common haplotypes, whereas the C2, C3, and DCHI variants were completely or almost completely (C2) homogeneous. There was a total genotypic agreement between the BbvI polymorphism and the presence/absence of the TF C3 variant, and the mutation that creates the BhvI site was found to lead to a G258S amino acid substitution.

C3-deficient guinea pigs possess a structurally altered, functionally inactive C3 variant and active C3 containing a functional thiolester

C3-deficient guinea pigs possess a structurally altered, functionally inactive C3 variant and active C3 containing a functional thiolester

Recurrent haemolytic uraemic syndrome and acquired hypomorphic variant of the third component of complement

In a girl with recurrent haemolytic uraemic syndrome (HUS), persistently low serum levels of C3 were found. Analysis of complement phenotype revealed a hypomorphic variant of C3 Fast in the patient (C3fS) and a normal heterozygous pattern in both parents and the brother (C3FS). Other complement aberrations in the patient were: the presence of a null gene for C4A and C4B and low serum levels of factor H. The father also had partial factor H deficiency. It is hypothesized that the hypomorphic C3 variant may predispose to recurrent HUS. In the acquired forms the role of uraemia in alteration of C3F should be considered.

Population studies of the c3 variant and esterase d polymorphism in the Japanese, and their determination from bloodstains and aged blood

Population studies of the c3 variant and esterase d polymorphism in the Japanese, and their determination from bloodstains and aged blood