Identification of complement factor H variants that predispose to pre‐eclampsia: A genetic and functional study

The objective of the study was to investigate the role of genetic variants in complement proteins in pre‐eclampsia.


| I N TRODUC TION
Pre-eclampsia (PE) occurs in approximately 3% of pregnancies, 1 accounting annually for over 50 000 maternal and 900 000 perinatal deaths worldwide. 2,3It is characterised by new-onset hypertension associated with proteinuria and/or end-organ dysfunction after 20 weeks of gestation.Disease manifestations include HELLP (Haemolysis, Elevated Liver enzymes, Low Platelets), kidney failure, seizures (eclampsia), stroke and cardiovascular complications.Despite it being a common pregnancy complication, the molecular basis for PE is incompletely understood and there is no treatment other than delivery.
As pregnancy represents an allogeneic-type mismatch between the fetus and the maternal immune system, multiple mechanisms are needed to maintain tolerance.In particular, the process of self-recognition must be carefully regulated during placentation to allow for fetal extravillous trophoblast invasion through the maternal decidua and myometrium to enable endothelial remodelling and vasodilation of the spiral arteries. 4The involvement of complement in adaptive immune response as well as the interaction between complement and natural killer (NK) cells are potential ways in which homeostatic balance and control over invasive placental trophoblast cells could be achieved. 5During pregnancy, complement assists in the clearance of placental fragments that enter the maternal circulation as a result of syncytiotrophoblast turn-over. 6ntegrin-type complement receptors 3 (CR3; CD11b/18) and 4 (CR4; CD11c/18) play important roles in the phagocytosis of complement iC3b-opsonised particles. 7One prevailing hypothesis is that improper clearance of such components, driven by an inadequately regulated complement cascade, may lead to deposition of debris in tissues and vascular walls, leading to an overly exuberant inflammatory response. 8Such unwarranted complement activation could provoke a maladaptive maternal immune attack against fetoplacental structures, leading to microthrombi and endothelial damage in PE, characteristic of a thrombotic microangiopathy (TMA). 9Complement activation at the maternal-fetal border may thereby result in disordered maternal-fetal tolerance.Lack of tolerance may be a basis for inadequate spiral artery remodelling, a key contributor to the pathogenesis of early-onset PE (diagnosis before 34 weeks of gestation). 10n overly exuberant inflammatory response driven by a dysregulated complement cascade has been implicated in a number of other diseases such as atypical haemolytic uremic syndrome (aHUS), C3 glomerulopathy (C3G), paroxysmal nocturnal haemoglobinuria (PNH) and age-related macular degeneration (AMD). 11The process in PE appears most analogous to that seen in aHUS, a complement-mediated TMA, characterised by endothelial injury leading to kidney failure, stroke and cardiovascular complications.Atypical HUS occurs due to loss-of-function mutations in complement regulators factor H, factor I or membrane cofactor protein (CD46) or gain-of-function mutations in complement proteins C3 or factor B. 12,13 Understanding the genetic basis has led to the development of anticomplement therapies that have revolutionised the management of PNH and aHUS.In susceptible women, pregnancy is a known trigger for aHUS. 14][17] Because of the similarity between PE and aHUS and the potential of complement overactivation to cause PE in a subset of high-risk patients, we investigated the role of genetic variants in one of the major complement regulators, factor H (FH) in a cohort of Finnish patients with PE. 9 An advantage of examining the Finnish founder population to study variants in complex diseases is that it is a more homogeneous population and thus disease-causing variants may be population-specific and occur at a higher frequency than in other, related European populations.Main outcome measures: Secretion, expression and ability to regulate complement activation were assessed for factor H proteins harbouring the mutations.Results: We identified five heterozygous rare variants in complement factor H (L3V, R127H, R166Q, C1077S and N1176K) in seven women with severe pre-eclampsia.These variants were not identified in controls.Variants C1077S and N1176K were novel.Antigenic, functional and structural analyses established that four (R127H, R166Q, C1077S and N1176K) were deleterious.Variants R127H and C1077S were synthesised, but not secreted.Variants R166Q and N1176K were secreted normally but showed reduced binding to C3b and consequently defective complement regulatory activity.No defect was identified for L3V.Conclusions: These results suggest that complement dysregulation due to mutations in complement factor H is among the pathophysiological mechanisms underlying severe pre-eclampsia.

K E Y W O R D S
complement system, factor H, genetics, pre-eclampsia: basic science 2 | M ET HODS

| Subjects and sequencing protocol
The Finnish Genetics of Pre-eclampsia Consortium (FINNPEC) study consisted of 1377 family trios (mother, father and infant) affected by PE or non-PE pregnancy, where biological samples and comprehensive clinical data were available to allow for in-depth analyses. 19n the FINNPEC cohort, PE was defined as hypertension and proteinuria occurring after 20 weeks of gestation.Hypertension was defined as systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg.Proteinuria was defined as the urinary excretion of ≥0.3 g protein in a 24-h specimen, or 0.3 g/l, or two ≥1 + readings on a dipstick in a random urine determination with no evidence of a urinary tract infection.The control group consisted of pregnant women who did not meet the criteria for PE.Early onset PE was defined as presentation of symptoms before 34 weeks of gestation and severe PE was diagnosed if at least one of the following criteria were fulfilled: blood pressure ≥160 mmHg systolic and/or ≥110 mmHg diastolic, proteinuria ≥5 g/day, other findings or symptoms indicating multi-organ and/or placental involvement, including headache and upper gastric pain.HELLP syndrome was diagnosed when at least two of the following criteria were met: lactate dehydrogenase ≥235 U/l, alanine aminotransferase ≥70 U/l, aspartate aminotransferase ≥70 U/L, platelets ≤100 E9/L.The subjects and methodology of this study have been described in detail previously (Table S1). 19,20For the current study, nulliparous or multiparous women with a singleton pregnancy and no history of chronic hypertension, diabetes or renal disease were considered eligible.All patients were self-declared white women.A jury consisting of a midwife and an obstetrician independently confirmed the diagnosis of PE.For the diagnosis, hypertension and proteinuria occurring after 20 weeks of gestation were required.Hypertension was defined as a systolic blood pressure of ≥140 mm Hg and/or a diastolic blood pressure of ≥90 mm Hg after 20 weeks of gestation.Proteinuria was defined as the urinary excretion of ≥0.3 g protein in a 24-h specimen or >0.3 g/L of urine, or two positive dipstick readings in the absence of a urinary tract infection.In Stage 1, we enrolled 487 non-obese (body mass index <30 kg/m 2 ) women with PE pregnancies and 187 non-PE controls from the FINNPEC cohort who fulfilled the required diagnostic criteria and had DNA available for genetic sequencing. 19In Stage 2, we enrolled 122 women with a history of PE (based on a clinician's diagnosis obtained through the comprehensive national Hospital Discharge Register), and 1905 parous controls with no such history from the national FINRISK study (FINRISK licence # 8/2016). 21National FINRISK study description and ethical approvals are available online: https://thl.fi/documents/32875 43/58085 77/FINRI SK_readm efirst_220920.pdf/aafab52b-a53b-d23c-73d7-15c55 81b24 fa?t=16007 69319413.For the association studies, genotypes from Stage 1 individuals were combined with the population cohort in Stage 2, providing a total sample size of 609 PE cases (487 + 122) and 2092 non-PE controls (187 + 1905).
In a previously described custom-made targeted exomic sequencing protocol, we combined Illumina sequencing libraries for capture and sequencing with Nimblegen sequence capture. 20,22

| Factor H quantification
ELISA was used to determine FH concentrations in third trimester patient sera.To quantify FH, Nunc Maxisorp plates were first coated with 100 microl portions of a monospecific mouse antibody against FH (196X, 5 microg/ ml).After an overnight incubation at 4°C, the plates were washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20 and blocked with 200 microl of PBS-Tween for 2 h.Duplicate serum samples in varying dilutions were added and incubated for 2 h at 37°C, whereafter the plates were washed with PBS.Polyclonal rabbit anti-human FH diluted at 1:2000 in PBS was added.After washing, IgG-HRP (horseradish peroxidase)-conjugated anti-rabbit IgG was added.After an incubation at 37°C for 1 h, the plates were washed with PBS and o-phenylenediamine dihydrochloride (OPD) substrate was added.The reaction was stopped with 120 microl of 0.5 M H 2 SO 4 and a spectrophotometer was used to measure the optical density of samples at 492 nm wavelength.FH concentrations were determined from comparison to standard samples with known amounts.

| Functional studies
The functional consequences of rare and unique variants observed in CFH were characterised using 293T cells transfected under serum-free conditions. 23Functional studies and surface plasmon resonance (SPR) interaction analyses with the Biacore X100 and Biacore 2000 instruments (GE Healthcare) were performed as previously described. 22,24ariants in CCP19 and CCP20 were purified as described, with the exception that they were eluted from a HisTrap column (GE Healthcare) at 285 mM NaCl as compared with variants in CCP2 and 3, which were eluted at 95 mM. 24umbering of variants includes the 18-amino acid signal peptide.Structural analyses were done in RCSB PDB. 25 Figure 3A is based on FH domains 1-4 in complex with C3b (PBD:2WII), 26 Figure 3B is based on structure of FH domains 18-20 (PBD:3SW0), 27 and Figure 3C is based on structure of FH CCP19-20 in complex with C3d (PBD:2XQW). 28

| Statistics
Sequence data were analysed in PLINK/Seq, Plink 29 and R programmes.Analyses of the significant associations were performed using Fisher's exact test.p-Values <0.05 were considered significant.Kaviar 30 and VEP Build 37 were used in additional annotations. 31In addition to an appropriate statistical probability test, odds ratios (OR) with 95% confidence intervals (95% CI) were calculated for all variants.

| Study approval
All subjects provided written informed consent for the study.The FINNPEC study protocol was approved by the coordinating Ethics Committee of the Hospital District of Helsinki and Uusimaa (permit number 149/E0/07).Patients and funders were not involved in study planning, data analysis or reporting of the results.

| R E SU LTS
Gene-based burden testing for rare and unique variants (minor allele frequency [MAF] <1%) in the FINNPEC cohort revealed that variation in CFH was associated with PE (non-adjusted P-value = 0.035, Bonferroni corrected P-value = 0.0003). 22We identified seven women with severe PE who carried five heterozygous rare variants in CFH (L3V, R127H, R166Q, C1077S and N1176K) (Table 1) among 609 cases.According to the registry-based health data, none of these patients developed aHUS during <10-year follow-up.
Two of these variants were novel (C1077S and N1176K), having not been described in the literature before.None of these five variants was identified in the 2092 controls.Of the seven women carrying CFH variants, six were diagnosed with early-onset PE.Two mothers developed HELLP syndrome (Figure 1A).Three of the seven were delivered by caesarean section.The mean pre-pregnancy body mass index (BMI) was in the normal range at 23.5 kg/m 2 .All seven women delivered infants that were small for gestational age (SGA, birthweight <−2 SD).
As assessed by western blotting, the variants R127H and C1077S were not expressed (Figure 1B,C).Western blotting for the lysates for both variants demonstrated intracellular accumulation, thus establishing that the proteins were synthesised but secretion in these heterozygous patients was reduced by 50% (not shown).These results were consistent with the reduced serum FH antigenic level in the patient carrying R127H, which showed that the FH level was decreased by 57.5% compared with normal human serum, which was used as reference (patient levels: approximately 0.21 g/L; normal range = 0.15-0.45g/L).R127H has been described previously in two brothers with membranoproliferative glomerulonephritis (MPGN) 32 and was shown to be associated with low antigenic levels.This variant has also been reported in a family with advanced AMD and was shown not to be secreted. 33,34The substitution of guanine to adenine (which results in the change from an R to H at the 127th amino acid) is predicted to be deleterious to the protein structure according to in silico prediction software programmes.
Serum for the patient carrying the C1077S variant was not available; however, structural analysis for C1077S indicated that the likely reason for decreased production was a lack of formation of a disulphide bridge, with a consequent change in the folding of the CCP18 domain (Figure 3B).
The variant R166Q has been previously reported in an AMD patient. 35R166Q was expressed in an equivalent amount to the wild type (WT) CCP1-4 but showed a ~10-fold reduced binding (64.4 versus 6.8 microM) to C3b (Figure 2A,B).The reduced binding to C3b leads to decreased cofactor activity, compared with the WT (Figure 2C).This decreased function is probably due to the loss of positive charge (owing to substitution of positively charged arginine by uncharged glutamine) in CCP3, which is critical to C3b binding (Figure 3A).Another notable difference between WT and R166Q was the pattern of cleavage of C3b to iC3b (Figure 2A,B).Normal cofactor activity produces a 41-kDa α-chain fragment and releases a 2-kDa fragment (the C3f fragment).The R166Q variant did not cleave the 2-kDa C3f fragment, thereby producing an incompletely cleaved α-chain fragment of 43 kDa (Figure 2C).Thus, both the efficiency and type of cleavage of the α-chain was affected by this mutation (Figure 2D,E).Structural analysis for R166Q demonstrated that this variant is located in a unique hypervariable loop of FH, which is significant for C3b binding.The variant N1176K was expressed equivalently to WT FH CCP18-20 (Figure 1C) and western blotting of the lysates did not show intracellular accumulation (not shown).Structural analysis for N1176K established that N1176 on the surface of FH CCP20 is adjacent to the C3d fragment.The change to 1176K by a missense mutation might alter the C3d binding surface (Figure 3C).By competing with Y1225 further across from N1176, the positively charged N1176K could form a new cation-π interaction with Y1177.Thereby, the replacement of this missense mutation N1176K might alter the FH C-terminal C3d binding surface.Indeed, the expressed recombinant protein demonstrated reduced binding to C3b and C3d (Figure 4), thus establishing that N1176K has decreased functional activity.
Variant L3V (rs139254423) was identified in three individuals and significantly associated with PE (P = 0.01, odds ratio 24.1150, 95% CI 1.25-467.20;MAF in cases = 0.25).However, the expression of variant L3V was comparable to WT (Figure 1A) and no functional defect was observed (not shown).These results were as consistent with normal serum FH antigenic level in two of three patients carrying this variant (serum was not available from the third patient).Soluble FLT1 (sFlt1) and placental growth factor (PlGF) levels in third trimester patient sera were available for two of the cases with the L3V variant.Both patients had a significantly elevated sFlt1 levels with a high sFlt1/PlGF ratio of 447 and 520.

DISCUS SION
Pre-eclampsia is a heterogeneous disease with multiple genetic and environmental risk factors.Although complement activation has been implicated in PE, 33,36,37 the aetiology of an overactive system has not been clearly delineated.

| Main findings
Gene-burden testing approach allows for association analysis between cases and controls by grouping rare protein-altering variants within a single gene.Using this method, we found factor H variants to be linked to severe pre-eclampsia. 22Our results identify maternal genetic variants in complement FH, the chief regulator of the alternative pathway of complement, in 1.4% of patients with PE.Four of these variants resulted in a dysfunctional FH, which in turn led to a dysregulated complement system. 38This is in line with recent findings of decreased serum levels of FH in PE in comparison with healthy pregnancy and 1.2% prevalence of CFH variants in PE or HELLP syndrome. 39,40We describe four missense variants within CFH gene that cause either a quantitative defect (decreased secretion as in R127H and C1077S) or a qualitative defect (normal secretion but defective function, as in R166Q and N1176K).Of note, the variant R127H has also been described in kidney and eye diseases (aHUS and AMD, respectively) known to be associated with complement overactivation. 32,33he variant R166Q, reported once in AMD, leads to an aberrant cofactor activity which is expected to affect both soluble C3b and C3b bound to a surface. 22,35The variant N1176K has not been described before and demonstrates reduced binding for both C3b and C3d, which likely affects the cleavage of C3b bound to self surfaces coated with sialic acids or polyanionic glycosaminoglycans.A rare variant within the signal peptide of FH (L3V) was also identified in three patients with severe PE.L3V alters the signal peptide sequence before the first 20 short consensus repeats (SCRs) that make up the FH molecule.However, the 3V protein appeared to be of the correct size with normal functional activity and the level of FH in two of our three patients with an available serum sample were normal, therefore the aetiology of severe PE in patients carrying this variant remains unclear. 4L3V is a rare variant classified as a variant of unknown significance with a minor allele frequency (MAF) of 0.024% in the genome aggregation database (gnomAD).There are six submissions listed in the ClinVar database, four of which were related to complement dysregulatory disorders: two in AMD, one in aHUS and one in membranoproliferative glomerulonephritis (MPGN).Our study, however, demonstrated that the L3V variant has a normal secretory profile and no apparent functional defects.Similarly, rare variant S58A in CFH with an MAF of 0.025% in gnomAD has been reported in a pregnant woman with SLE who developed PE.Despite extensive investigations, no functional defect was found. 22,41These data strongly point to rare variants in FH predisposing pregnant women to an increased risk of developing PE.However, the underlying mechanisms remain unclear and warrant further investigation. 22,41otably, among the seven patients with CFH rare variants, 28% had HELLP syndrome (a rare life-threatening complication of PE) compared with 7.4% in patients with PE in the FINNPEC cohort. 42Pre-existing kidney disease or autoimmune conditions were not reported for any of the patients and proteinuria levels were typical for severe PE.Therefore, patients with deleterious CFH variants represent a TMA-like severe PE phenotype, rather than an underlying kidney disease.
Our results establish that regulation of the maternal complement system by FH is essential for a healthy pregnancy, and mutations affecting this process predispose to PE.These patients likely represent a high-risk group who present with severe PE and HELLP syndrome, validating a potential subphenotype of PE with TMA features. 9These data also lay the groundwork for future studies to assess the association of PE with defects in complement proteins in other cohorts and provide further support for treatment of selected severe PE/HELLP cases with complement-specific therapies such as eculizumab.Our results support a link to the FH signal peptide variants, but the functional mechanisms have yet to be revealed.Furthermore, replication in a second population is needed, preferably including non-European patients.The role of fetal complement genes in the predisposition to PE also remains to be explored.

AU T HOR C ON T R I BU T ION S
AIL performed the genetic studies with MT, ED and MD, and wrote the first draft of the paper.ZR and MT performed the functional studies for the discovered variants.FINNPEC provided samples for the study and bers and RB reviewed and approved the paper.MP and KA provided samples from the Finrisk cohort.JES designed the original genotyping platform with JPA and MT.HL is the principal investigator responsible for FINNPEC samples and data and designed the genetic study with JPA.JPA, AJ and SM supervised the data acquisition and helped to draft the paper and figures.All co-authors read and approved the paper.

F I G U R E 2
Functional evaluation of the WT FH CCP1-4 and the variant R166Q.(A,B) Affinity of R166Q for C3b by surface plasmon resonance (SPR).R166Q had decreased affinity for human purified C3b in comparison with the WT CCP1-4.(C-E) The fluid-phase C3b cofactor activity of the variant R166Q with purified human Factor I (FI) was assessed by cleavage of purified C3b to iC3b and was compared with the WT FH CCP1-4.The percentage of the α′ chain remaining and the generation of the α1 fragment indicate cleavage of C3b to iC3b.The kinetic analyses of cofactor activity were conducted at 0, 5, 10 and 20 min.Lane 9 represents a positive control employing purified human FH and Lane 10 represents a negative control employing concentrated cell media in the presence of both purified human FI and C3b.The cleavage rate was measured by densitometric analysis of the remaining α′ chain and by the generation of α1 relative to the β chain.This assay was repeated three times.CCP, complement control protein (repeats); FH, factor H; WT, wild type.Residue numbering includes the 18 amino acids of the signal peptide (SP).

F I G U R E 3
Structural analysis of FH variants.(A) Structural analysis of R127H and R166Q.Complement C3b (brown and green) in complex FH CCP1-4 (purple) demonstrating where variants R127H and R166Q are located.R166 is in the unique hypervariable loop of domain 3 of FH, which has been shown to interact with C3b.(B) Factor H CCP18-20 with variants C1077S and N1176K highlighted.(C) Factor H CCP19-20 (orange) in complex with C3d (green) shown in cartoon.N1176 (purple) is on the surface of FH CCP20 and is adjacent to the C3d fragment.CCP19-20 has a contiguous binding surface for C3d including Y1177.Y1225 is spatially on the opposite side of N1176 and forms a cation-p interaction with Y1177.CCP, complement control protein (repeats); FH, factor H; WT, wild type.Residue numbering includes the 18 amino acids of the signal peptide (SP).
18 Medical Foundation; University of Helsinki Funds; Special State Subsidy for Health Research at Helsinki University Hospital, Grant/Award Number: TYH2022315; Sakari and Päivikki Sohlberg Foundation; Novo Nordisk Foundation; Signe and Ane Gyllenberg Foundation; Foundation for Paediatric Research Clinical and pathological characteristics of patients with rare missense mutations in CFH.