Structural and Functional Characterization of the Product of Disease-Related Factor H Gene Conversion

Numerous complement factor H (FH) mutations predispose patients to atypical hemolytic uremic syndrome (aHUS) and other disorders arising from inadequately regulated complement activation. No unifying structural or mechanistic consequences have been ascribed to these mutants beyond impaired self-cell protection. The S1191L and V1197A mutations toward the C-terminus of FH, which occur in patients singly or together, arose from gene conversion between <i>CFH</i> encoding FH and <i>CFHR1</i> encoding FH-related 1. We show that neither single nor double mutations structurally perturbed recombinant proteins consisting of the FH C-terminal modules, 19 and 20 (FH19-20), although all three FH19-20 mutants were poor, compared to wild-type FH19-20, at promoting hemolysis of C3b-coated erythrocytes through competition with full-length FH. Indeed, our new crystal structure of the S1191L mutant of FH19-20 complexed with an activation-specific complement fragment, C3d, was nearly identical to that of the wild-type FH19-20:C3d complex, consistent with mutants binding to C3b with wild-type-like affinity. The S1191L mutation enhanced thermal stability of module 20, whereas the V1197A mutation dramatically decreased it. Thus, although mutant proteins were folded at 37 °C, they differ in conformational rigidity. Neither single substitutions nor double substitutions increased measurably the extent of FH19-20 self-association, nor did these mutations significantly affect the affinity of FH19-20 for three glycosaminoglycans, despite critical roles of module 20 in recognizing polyanionic self-surface markers. Unexpectedly, FH19-20 mutants containing Leu1191 self-associated on a heparin-coated surface to a higher degree than on surfaces coated with dermatan or chondroitin sulfates. Thus, potentially disease-related functional distinctions between mutants, and between FH and FH-related 1, may manifest in the presence of specific glycosaminoglycans

Homozygous <i>CFHR3/CFHR1</i> deletion frequencies in UK and HGDP-CEPH worldwide populations.

<p>The P value derived using Fisher's exact test compare either the genotype frequencies (del⁺⁺/del⁺⁻/del⁻⁻) or the frequency of homozygous <i>CFHR3/CFHR1</i> deletion (del⁺⁺) in HGDP-CEPH populations with that of the UK population or all other populations combined.</p

Table <b>4.</b> Reported population frequencies of the <i>CFHR3/CFHR1</i> deletion.

<p>MLPA, multiplex ligation-dependent probe amplification.</p><p>WB, western blotting.</p><p>PCR, polymerase chain reaction.</p><p>CEU, Utah residents with Northern and Western European ancestry from the CEPH collection.</p><p>CHB, Han Chinese in Beijing, China.</p><p>CHD, Chinese in Metropolitan Denver, Colorado.</p><p>GIH, Gujarati Indians in Houston, Texas.</p><p>JPT, Japanese in Tokyo, Japan.</p><p>LWK, Luhya in Webuye, Kenya.</p><p>MEX, Mexican ancestry in Los Angeles, California.</p><p>MKK, Maasai in Kinyawa, Kenya.</p><p>TSI, Toscans in Italy.</p><p>YRI, Yoruba in Ibadan, Nigeria.</p><p>ASW, African ancestry in Southwest USA.</p

Clinical characteristics of non-autoimmune preeclampsia patients with complement regulatory protein mutations.

<p>All patients were white.</p>a<p>Previous pregnancy preeclampsia delivered at 36 wk.</p>b<p>Pregnancy complicated by sphenoid sinus thrombosis; thrombophilia evaluation negative; four normal previous pregnancies; history of idiopathic thrombocytopenia purpura; mother with SLE.</p>c<p>Oligohydramnios.</p>d<p>Twins.</p><p>GA, gestational age.</p

Clinical characteristics of autoimmune patients with complement regulatory protein mutations.

<p>Patients were white, except the patient with MCP K32N, who was black. Characteristics of preeclampsia <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.1001013#pmed.1001013-ACOG1" target="_blank">[16]</a>.</p>a<p>Severe preeclampsia with visual disturbance.</p>b<p>Preeclampsia with oligohydramnios and intrauterine growth restriction (<5th percentile).</p>c<p>Severe preeclampsia, HELLP syndrome.</p><p>GA, gestational age.</p

Preeclampsia in autoimmune PROMISSE patients.

a<p>The PROMISSE Study pregnancy was the first pregnancy in 40% of the SLE patients (with and without APL Ab) and in 9% for APL Ab patients without SLE. The APL Ab patients differed from SLE patients with regard to multiparity because pregnancy loss is a criterion for testing for APL Ab.</p>b<p>Three patients had preeclampsia in the PROMISSE Study and also had preeclampsia in previous pregnancies.</p

Complement activation in preeclampsia.

<p>Black and blue arrows indicate triggering of complement cascades by autoantibodies or damaged self-tissue; green arrows, deposition of C3b on a target sets in motion the powerful amplification loop of the alternative pathway; red arrows, effector activities of complement are generated by C3b deposition and C3a release and the downstream mediators C5b-9 and C5a. Regulation of feedback loop of alternative pathway on the placental trophoblasts (or endothelial cells) occurs through limited proteolytic cleavage of C3b to generate iC3b. This reaction is carried out by a serine protease, factor I (FI), along with membrane cofactor protein (MCP) or CFH (FH). Because SLE and APS are characterized by autoantibodies that trigger the classical pathway, defective regulation of C4b (a component of the classical pathway C3 convertase) by MCP is also likely to influence the severity of tissue injury and risk for preeclampsia. If regulators such as MCP, CFI (FI), or CFH (FH) are dysfunctional, excessive complement activation occurs. This may result in placental damage, thrombobosis and release of antiangiogenic factors, culminating in preeclampsia.</p

C3b and C4b binding and cofactor activity of K32N compared to wild type MCP.

<p>(A) C4b and C3b binding. CHO cell lysates from transient transfections were incubated with C4b- or C3b-coated wells and binding was assessed using a polyclonal antibody to MCP in an ELISA format. CHO, a mock transfected control. Data represent the mean ± standard deviation at each concentration from three independent experiments. (B) Cofactor activity for CFI-mediated cleavage of C4b and C3b. Cell lysates containing 25 pg of MCP (wild-type or K32N) were incubated with biotinylated C4b or C3b in the presence of purified human CFI in 25 mM NaCl and cleavage was assessed by Western blot. C4b and C3b peptide chains and cleavage fragments are identified. For C4b cofactor activity, C4d fragment generated by cleavage of C4b after 90 min was quantified by densitometric scanning, compared with quantity of β chain, and normalized to wild type. For C3b cofactor activity, α1 fragment generated by cleavage was compared to β chain by densitometry, and normalized to wild type. Data represent the mean ± standard deviation of three independent experiments. (C) Representative Western blots of MCP activity for CFI-mediated cleavage of C4b and C3b. Left panel: MCP K32N has deficient C4b cofactor activity at 20 and 90 min. The C4d fragment generated by cleavage of the α′ chain is not visible at 20 min and is diminished relative to wild type at 90 min. The boxed inserts represent 5-fold longer exposure of the blot. The CHO negative control was a nonadjacent lane (broken line). Right panel: MCP K32N has similar C3b cofactor activity to wild type. Equal amounts of α1 fragments are generated after 20 and 90 min. The CHO negative control was a nonadjacent lane (broken line). In two additional experiments, similar results were obtained in cofactor assays at 150 mM NaCl concentration (unpublished data).</p

Effects of rare kidney diseases on kidney failure: a longitudinal analysis of the UK National Registry of Rare Kidney Diseases (RaDaR) cohort

Individuals with rare kidney diseases account for 5-10% of people with chronic kidney disease, but constitute more than 25% of patients receiving kidney replacement therapy. The National Registry of Rare Kidney Diseases (RaDaR) gathers longitudinal data from patients with these conditions, which we used to study disease progression and outcomes of death and kidney failure.People aged 0-96 years living with 28 types of rare kidney diseases were recruited from 108 UK renal care facilities. The primary outcomes were cumulative incidence of mortality and kidney failure in individuals with rare kidney diseases, which were calculated and compared with that of unselected patients with chronic kidney disease. Cumulative incidence and Kaplan-Meier survival estimates were calculated for the following outcomes: median age at kidney failure; median age at death; time from start of dialysis to death; and time from diagnosis to estimated glomerular filtration rate (eGFR) thresholds, allowing calculation of time from last eGFR of 75 mL/min per 1·73 m2 or more to first eGFR of less than 30 mL/min per 1·73 m2 (the therapeutic trial window).Between Jan 18, 2010, and July 25, 2022, 27 285 participants were recruited to RaDaR. Median follow-up time from diagnosis was 9·6 years (IQR 5·9-16·7). RaDaR participants had significantly higher 5-year cumulative incidence of kidney failure than 2·81 million UK patients with all-cause chronic kidney disease (28% vs 1%; p Background Methods Findings Interpretation Funding</p