Common Haplotypes within the Chromosome 1q31.3 Region Determine Systemic Concentrations of the Entire Complement Factor H Protein Family Open Access

The complement system is a pivotal part of our innate immunity and requires optimal regulation to prevent unwarranted attack on host cells and tissues. The factor H (FH) protein family is a group of soluble plasma proteins that regulate the alternative pathway of complement activation [1]. The family is comprisedseven members that share a high degree of amino acid sequence similarity. Apart from FH, this family includes FHL-1, a splice variant of FH, and five FH paralogs known as the FH-related proteins (FHR-1 to -5) [2, 3]. Each FHR is encoded by its own gene in tandem and in proximity across the CFH locus on chromosome 1q31.3. Alternative splicing of CFHR4 has been described, resulting in FHR-4A and FHR-4B, but the latter has not been detected in circulation [4, 5]. Furthermore, FHR-1, -2, and -5 form dimers, with FHR-1 and -2 forming both homo- and heterodimers, whereas FHR-5 only forms homodimers in circulation [6, 7]. Although extrahepatic expression has been described for several family members, with its potential impact on local complement regulation, FH, FHL-1, and the FHR proteins are primarily produced in the liver, with blood being the main matrix [8‒13].

FH consists of twenty complement control protein (CCP) domains, while FHL-1 only contains the first seven N-terminal CCPs of FH, with the addition of the four unique amino acid sequence SFTL at its C-terminus [14, 15]. Complement regulation by FH and FHL-1 is mediated through the first four N-terminal CCP domains. To enable complement regulation on cell surfaces, FH and FHL-1 bind to specific ligands on host cells via CCP6–7, while for FH alone, additional binding occurs through CCP19–20 [16]. The FHR proteins have varying numbers of CCP domains, ranging from four (FHR-2) to nine (FHR-4A and FHR-5). Furthermore, the FHRs seem to lack CCP domains capable of directly regulating complement, but all have CCP domains corresponding with CCP6–7 and/or CCP19–20 of FH. This has led to the current model that FHRs act as antagonists of FH and possibly FHL-1 on cell surfaces [17]. This competition could hinder complement inhibition by FH or FHL-1, thereby potentially contributing to further complement activation rather than limiting it [18].

Genetic variation within the CFH locus has been associated with a wide range of diseases, including atypical haemolytic uraemic syndrome (aHUS), age-related macular degeneration (AMD), meningococcal disease, and C3-glomerulopathy among others [19‒22]. Extensive genetic studies investigating common single nucleotide polymorphisms (SNPs) or complete gene deletions (CFHR3/CFHR1 and CFHR1/CFHR4) within the CFH locus have shown that many of these SNPs are in strong linkage disequilibrium, resulting in the identification of four major haplotypes (H1–H4, online suppl. Table S1; for all online suppl. material, see https://doi.org/10.1159/000545342) [23‒26]. These haplotypes capture more than 99% of control- and case-associated chromosomes and account for more than 90% of the genetic variability at the CFH locus within the Western population [23, 27]. Haplotype H1 is marked by the SNP rs1061170 (Tyr402His) in CCP7 of FH and FHL-1, leading to altered ligand binding, and is associated with a significant increase in AMD risk but provides protection for aHUS [24]. H2 is characterized by rs800292 (Val62Ile) in FH/FHL-1 CCP1, which results in modestly higher affinity for C3b, enhancing its cofactor activity and conferring protection for both AMD and aHUS among others [24, 25, 28]. Haplotype H3 is characterized by a cluster of genetic variants (rs3753394, rs3753396, rs1065489) in CFH and is associated with an increased risk for aHUS, protection for meningococcal disease and a neutral effect for AMD [19, 20, 29, 30]. Lastly, haplotype H4 is hallmarked by the deletion of both CFHR3 and CFHR1, which is deemed protective to AMD, IgA nephropathy, and C3 glomerulopathy, but a risk factor for the development for autoimmune aHUS and systemic lupus erythematosus [25, 26, 31‒35]. These haplotypes seem to encompass the entire CFH locus, with H1 and H2 including eQTL genetic variants in CFHR4 that significantly influence FHR-4 levels [21]. Likewise, H3 comprises variants within CFHR3, resulting in altered expression levels of FH and FHR-3 [29]. However, these studies commonly investigate a single or a few FH protein family members at a time, often within a case/control cohort. As such, a complete overview on how the four common haplotypes at the chromosome 1q31.3 locus are reflected in systemic levels of the entire FH protein family in a healthy population is lacking. This in part has been greatly hampered by lack of specific reagents and assays to specifically detect all FH family members, more precisely, assays to measure FHR-2 and FHL-1.

With the recent development of standardized ELISA kits for FHR-2, -3, -4 and FHR-5, and the here described first FHL-1-specific ELISA, we can now measure all FH protein family members, including all the distinct dimer species observed in vivo within one individual [36, 37]. This has enabled us to establish systemic reference levels for each FH family member found within a cohort of healthy individuals. Furthermore, we show distinct protein expression levels for each family member across the four common CFH haplotypes. As the FHR proteins are hypothesized to locally downregulate FH and FHL-1 function, impacting the balance of complement activation and regulation, understanding how qualitative and quantitative genetic variants influence the FH protein family is essential. This is part of the complotype; the aggregate of all genetic variation within the complement system that dictates its overall activity and regulation [38]. The obtained reference intervals and genetic determinants support further investigations into this complex and interlinked protein family and can be particularly valuable in helping to understand how several of these haplotypes confer either risk or protection in complement-mediated diseases.

Plasma (Citrate: VACUETTE^® 9NC coagulation sodium citrate 3.2% [Cat# 455322, Greiner Bio-One, Kremsmünster, Austria]; heparin: VACUETTE^® Sodium Heparin [Cat# 455051, Greiner Bio-One]; and EDTA plasma: VACUETTE^® K3EDTA [Cat# 455036, Greiner Bio-One] and/or serum [clot activator: silica particles, VACUETTE^®, Cat# 455092, Greiner Bio-One]) samples were obtained between 2010 and 2024 (Sanquin Research, Amsterdam, The Netherlands) from anonymous healthy volunteers with written informed consent, adhering to the Dutch regulations. Serum and plasma samples were centrifuged at 1,600 g for 10 min at 4°C to collect serum or plasma. Before collecting the serum, blood was allowed to clot for 1 h at room temperature. For each donor, the peripheral blood mononuclear cell fraction of an EDTA sample was collected for DNA extraction using the QIAamp^® DNA blood Mini Kit (Qiagen, Hilden, Germany) following manufacturer’s instructions. Of eighteen donors, EDTA plasma samples were collected monthly following the same protocol. All samples were aliquoted and stored at −80°C.

For all ELISAs, incubation steps were performed at room temperature while shaking. After each incubation step, plates were washed five times with PBS + 0.02% (v/v) Tween^®-20 (PT) using a BioTek^® 405 LSRS plate washer (BioTek Instruments, Winooski, VT, USA). Absorbance was measured at 450 nm using a Synergy^™ 2 plate reader (BioTek Instruments) and corrected for background absorbance at 540 nm. For each step, an end volume of 100 µL was used unless stated otherwise. Median levels (µg/mL) are reported unless stated otherwise.

Levels of FH, FHR-4A, and dimers of FHR-1 and -2 were quantified as previously described using in-house developed protocols [5, 36, 39]. ELISA kits and reagents for the measurement of FHR-2 (cat# HK3004), -3 (cat# HK3005), -4 (cat# HK3006), and FHR-5 (cat# HK3007) were provided by Hycult^® Biotech (Uden, The Netherlands), and assays were performed following manufacturer’s protocols. Development of these kits is described in van Rossum et al. [37].

For the measurement of FHL-1, an FHL-1 specific polyclonal antibody (aCTM119, University of Utah, John A. Moran Eye Center, Salt Lake City, USA) was coated (1.92 µg/mL in 0.1 m carbonate-bicarbonate buffer, pH 9.6) on Nunc MaxiSorp^™ F96 well plates (Cat# 437111, Thermo Fisher Scientific). Samples were diluted in high performance ELISA buffer (HPE, Cat# M1940, Essange, Amsterdam, The Netherlands) and incubated for 90 min. Next, bound FHL-1 was detected using the commercial biotinylated mAb OX-24 (0.125 µg/mL in HPE, Cat# AB_2539125, Thermo Fisher Scientific) before incubation with 0.01% (v/v) strep-poly-HRP (in PT0.1%, Cat# M2032, Essange) for 30 min. Lastly, plates were developed for 5 min using chemiluminescent detection (SuperSignal^™ ELISA Pico Chemiluminescent Substrate, Cat# 37069, Thermo Fisher Scientific) before measuring luminescence by a Synergy^™ 2 plate reader (BioTek^® Instruments). All steps were performed with a volume of 50 µL per well. Protein levels are expressed in µg/mL and were calculated using a calibrated standard curve of a normal human serum pool of more than 400 healthy donors as shown in online supplmenetary Figure S2a (kindly provided by Sanquin Diagnostic Services, Amsterdam, The Netherlands).

The genetic background (online suppl. Table S2) of all donors was previously determined using multiplex ligation-dependent probe amplification (MLPA) and/or next generation sequencing (NGS) [36]. In short, copy number variation (CNV) in the CFH locus (Salsa^® MLPA probe mix P236-A3 and/or -B1 CFH region, MRC Holland, Amsterdam, The Netherlands) and the presence of two SNPs (rs1061170 and rs4085749, in-house developed synthetic probes, online suppl. Table S3), were determined by MLPA. Additionally, the CFH region of 77 healthy donors was sequenced using a custom complement panel (Sanquin Complement Panel, Thermo Fisher Scientific) [40]. Both the preparation of the DNA library and the sequencing were performed according to manufacturer’s protocols using an Ion Torrent system (Ion Chef^™ and Ion S5^™ System, Thermo Fisher Scientific). Sequence data were analysed using Ion Reporter software workflow 5.16 (Thermo Fisher Scientific). For all donors, haplotypes (H1–H4) were determined by assessing the CNV of CFHR3/CFHR1 and analysing multiple SNPs (rs1061170, rs1410996, rs4085749) spanning the CFH locus, as described by Hughes et al. [25] (2006) and Bernabéu-Herrero et al. [26] (2015) (online suppl. Table S1). For 147 donors, haplotypes had previously been determined using the SALSA^® MLPA probe mix P236-A3 ARMD mix-1 from MRC Holland, which detects the presence of rs1061170, rs1410996 and determines the CFHR3/CFHR1 CNV. Since the current probe mix P236-B1 lacks SNP detection, used for the remaining 54 donors, in-house synthetic probes were used to detect rs1061170 and rs4085749 via MLPA and rs1410996 via PCR (online suppl. Table S3).

Data and statistical analysis were conducted using GraphPad Prism version 10.3.1 for Windows (GraphPad Software, San Diego, California USA). Before testing significance, a Shapiro-Wilk test was performed to test for normal distribution. The Friedman, the Mann-Whitney test, the Welch’s t test, the Kruskal-Wallis, the Brown-Forsythe and Welch ANOVA, and the one-way ANOVA test were used to assess significant differences, with p values below 0.05 indicating statistical significance. Where appropriate, correction for multiple testing was applied. The nonparametric Spearman correlation test was used to evaluate correlations.

To determine the robustness of the systemic levels of the FH protein family, nine males and nine females with a median age of 61.0 years (CI = 59.0–62.0 years) were followed over the course of 1 year (Table 1a). To mitigate any potential impact of age and sex on protein levels, the eighteen adults were specifically selected based on sex and age similarity. Next, serum and plasma (EDTA, citrate, heparin; CHES panel) samples were obtained from 104 blood donors to explore the potential matrix influence on protein levels (Table 1b). This group consisted of 68% female donors (median age = 48.5 years; CI = 43.0–52.0) and 32% male donors (median age = 52.5 years; CI = 35.0–59.0), with a median age of 50.5 years (CI = 43.0–53.0). Lastly, for the determination of systemic reference intervals of the FH protein family, serum samples were collected from 201 Dutch healthy individuals as part of a previous study (Table 1c) [36]. Among these individuals, 64% were female, with a median age of 43.0 years (CI = 41.0–46.0), while 36% were male, with a slightly higher median age of 50.0 years (CI = 41.0–53.0).

Eighteen donors were followed over the course of 1 year, during which multiple blood samples were collected to study the longitudinal stability of protein levels (Fig. 1). Quantification of all FH family members revealed consistent systemic levels, indicating the timing of sample collection has no significant impact on protein levels in healthy volunteers.

To investigate the potential influence of sample matrix on the levels of the FH protein family, paired serum and plasma (citrate, heparin, EDTA) samples were collected from 104 donors (Table 1b). Results were expressed as a percentage of recovery compared to serum, which was used as reference (Fig. 2). Absolute levels of the FH protein family in serum and plasma are summarized in Table 2 and presented in online supplementary Figure S1. Results for FH and the FHR proteins showed a consistent median protein recovery well within the expected range of assay variability (85–115%) with 93.6–105.2% and 96.6–109.0% for EDTA and heparin plasma, respectively. With an overall median recovery between 83.3 and 92.9%, protein in citrate plasma for FH (p < 0.0001) and FHR-1/1 (p = 0.040) was statistically significant lower, with similar trends for the other FHR proteins. This was likely attributed to the sodium citrate solution present in the collection tube, diluting the blood when drawing the sample (Fig. 2). While significant differences were observed with paired analysis between serum and EDTA (online suppl. Fig. S1), it is unclear whether these differences for FH and the FHRs are biologically relevant since approximately 87.0% of the donors were within the range of assay variability (Fig. 2). Furthermore, strong correlations were observed for all proteins among the different matrices (Table 2).

Like FH and the FHRs, the potential influence of sample matrix on the levels of FHL-1 was determined. In contrast to FH and the FHRs, FHL-1 displayed a significant higher median percentage of protein recovery in plasma compared to serum, with 120.1% (p < 0.0001), 115.0% (p < 0.0001), and 106.0% (p < 0.0001) for EDTA, heparin, and citrate plasma, respectively (Fig. 2). With most of the donors (EDTA = 70.0%, heparin = 51.0%) falling outside the range of assay variability (85–115%), results suggest a biological relevant matrix influence on levels of FHL-1, with coagulation being a potential explanation. To further investigate this, citrate plasma samples of 25 donors were recalcified to allow activation of the coagulation cascade. After clot formation, levels of FHL-1 were again quantified (Fig. 3a). Results showed similar levels of FHL-1 in recalcified citrate plasma and serum. In contrast, levels of FHL-1 in recalcified citrate were significantly lower (p = 0.0007) compared to its original citrate sample, underscoring the potential involvement of FHL-1 in coagulation. To validate this observation, the same principle was repeated for thirteen EDTA samples, showing similar results. In contrast, this effect of clot formation in citrate plasma on levels of FHL-1 was not observed for FH (Fig. 3c).

In this study, we established systemic in vivo reference levels of the entire FH protein family (Fig. 4a; Table 3). We report median levels of FH (279.00 µg/mL, CI = 263.70–291.30) and the FHRs (FHR-1/1 = 12.45 µg/mL, CI = 11.36–13.00; FHR-1/2 = 4.34 µg/mL, CI = 4.10–4.64; FHR-2/2 = 1.21 µg/mL, CI = 1.02–1.37; FHR-2 = 1.35 µg/mL, CI = 1.22–1.49; FHR-3 = 0.71 µg/mL, CI = 0.63–0.78; FHR-4 = 2.50 µg/mL, CI = 2.22–2.80; FHR-5 = 1.36 µg/mL, CI = 1.31–1.43) in serum of 201 Dutch healthy volunteers. Although to date FHR-4B has not been detected in human circulation, the FHR-4 ELISA used in this study was designed to measure both variants [37]. When quantifying both FHR-4 and FHR-4A levels in the same sample, similar absolute levels and a strong correlation were observed, supporting FHR-4A is likely the only form of FHR-4 present in circulation (online suppl. Fig. S2c, FHR-4 = 2.54 µg/mL, CI = 2.32–2.84; FHR-4A = 2.58 µg/mL, CI = 2.22–2.75; r_s = 0.97, p < 0.0001) [5]. Lastly, using an FHL-1 specific ELISA (online suppl. Fig. S2a), we report levels of FHL-1 with a median serum concentration of 0.96 µg/mL (CI = 0.92–1.01, 23.28 nm, molecular weight 43 kDa, Table 3).

When stratifying on sex (online suppl. Fig. S3a; Table 3), only levels of FHL-1 were significantly influenced with higher levels in females (1.03 µg/mL; CI = 0.96–1.07) compared to males (0.86 µg/mL; CI = 0.82–0.92, p = 0.0007). Additionally, levels of FH (r_s = 0.34, p = 0.0055) and FHR-1/1 (r_s = 0.34, p = 0.0052) in males and FHL-1 (r_s = 0.30, p = 0.0010) in females showed a weak positive correlation with age (online suppl. Fig. S3b; Table 3).

As the FHR proteins are suggested to compete with FH for binding to ligands, the molar ratio between FH and the FHRs may hold more biological significance than absolute levels when studying this protein family. Consequently, the molar ratio between FH and FHL-1 or the FHRs was calculated (Table 3). Results showed a considerable variation in mean ratio among the FHR proteins, with FHR-2/2 and -3 exhibiting the largest mean ratio (FH:FHR-2/2 = 141.1, CI = 100.5–181.1; FHR-3 = 131.7, CI = 118.4–145.00) and FHR-1/1 the smallest ratio (FH:FHR-1/1 = 5.2; CI = 5.1–5.9). As levels of FHL-1 were significantly influenced by coagulation, resulting in lower serum levels, the molar ratio between FH and FH family members was also determined in the different plasma types (online suppl. Fig. S4). As expected, the molar ratio for FH:FHL-1 was significantly higher in serum compared to the different plasma types (serum vs. EDTA and citrate plasma: p < 0.0001, serum versus heparin plasma: p = 0.0011). Also, the mean ratio between FH and FHR-5 was significantly higher in serum versus EDTA (p = 0.014) and heparin (p = 0.024) plasma.

Next, we investigated whether levels of the individual family members correlated with each other (Fig. 4b). A weak correlation was observed between levels of FH and FHR-5 (r_s = 0.37, p < 0.0001). Since FHR-1 and FHR-2 form dimers, correlations were observed between levels of all dimer configurations (FHR-1/1 vs. -1/2: r_s = 0.51, p < 0.0001; FHR-1/2 vs. -2/2: r_s = 0.67, p < 0.0001; FHR-1/2 vs. -2: r_s = 0.80, p < 0.0001; FHR-2/2 vs. -2: r_s = 0.89, p < 0.0001). Interestingly, levels of FHR-4 and -5 both showed a moderate correlation with FHR-2 (FHR-2 vs. -4: r_s = 0.45, p < 0.0001; FHR-2 vs. -5: r_s = 0.40, p < 0.0001) and both its derived dimers FHR-1/2 (FHR-1/2 vs. -4: r_s = 0.43, p < 0.0001; FHR-1/2 vs. -5: r_s = 0.39, p < 0.0001) and -2/2 (FHR-2/2 vs. -4: r_s = 0.40, p < 0.0001; FHR-2/2 vs. -5: r_s = 0.36, p < 0.0001). Additionally, levels of FHR-1/1 and -1/2 correlated with FHR-3 (FHR-1/1 vs. -3: r_s = 0.59, p < 0.0001; FHR-1/2 vs. -3: r_s = 0.38, p < 0.0001), explained by the combined CFHR3 and CFHR1 deletion. Lastly, although FHL-1 is a splice variant of FH, levels of FHL-1 showed no correlation with FH (r_s = −0.02). Interestingly, levels of FHL-1 did show a moderate correlation with FHR-3 (r_s = 0.60, p < 0.0001), irrespective of CFHR3 copy number status (CFHR3 CNV = 1: r_s = 0.53, p < 0.0001; CFHR3 CNV = 2: r_s = 0.62, p < 0.0001, online suppl. Fig. S2d).

To explore how genetic variants may affect protein levels, we stratified 187 donors on CFH haplotypes using a combination of MLPA, NGS, and PCR (online suppl. Table S1). Among them, 58 donors (31%) were homozygous for one of the four major CFH haplotypes. Five donors were identified as carriers of the less common H5 haplotype; however, none were homozygous. In addition, five individuals could not be stratified in one of the four major haplotypes and four donors had either deletions and/or duplication events of CFHR genes. Therefore, these individuals were excluded in further analyses.

We first investigated to influence of homozygous diplotypes on levels of the FH protein family (Fig. 5). A minor positive effect in H1 on levels of FHR-1/1, -1/2, -2, -4, and FHR-5 was observed. In contrast, H2 is associated with lower levels of FHR-2 and its dimers (FHR-2: H2 vs. H1 and H4: p < 0.0001; FHR-1/2: H2 vs. H1: p < 0.0001; FHR-2/2: H2 vs. H1 and H4: p < 0.0001) and with lower levels of FHR-4 (H2 vs. H1: p = 0.0001, H2 vs. H3: p = 0.037) and FHR-5 (H2 vs. H1: p = 0.019) as is shown in Figure 5a and b. Interestingly, in H3, levels of FHR-3 (H3 vs. H1: p = 0.041; H3 vs. H2: p = 0.0011) and FHL-1 (H3 vs. H1: p = 0.024; H3 vs. H2: p = 0.037; H3 vs. H4: p = 0.047) were elevated but showed decreased levels of FH (H3 vs. H4: p = 0.0036). As expected, haplotype H4, which involves the deletion of CFHR3/CFHR1, resulted in complete lack of FHR-1/1, -1/2, and FHR-3. Furthermore, this haplotype also showed elevated levels of FHR-2/2 (H4 vs. H1: p = 0.033; H4 vs. H3: p = 0.0093), caused by the absence of FHR-1, resulting in no formation of FHR-1/2 heterodimers and all FHR-2 being present as FHR-2/2 homodimers. Surprisingly, baseline levels of FHR-2 and FH were both elevated in individuals carrying the H4 haplotype (FH: H4 vs. H2: p = 0.017). A summary of median protein concentration per diplotype for all FH protein family members is provided in Table 4.

The influence of different haplotypes on protein levels remains notable when analysing heterozygous diplotypes (Fig. 5a; online suppl. Fig. S5; Table 4). Due to the deletion of CFHR3/CFHR1 in haplotype H4, mixed diplotypes containing H4 exhibit reduced levels of FHR-1/1. Similarly, FHR-3 levels in H1/H4 and H2/H4 were affected by H4, though the negative effect of H4 was neutralized by elevated FHR-3 levels in H3 within the H3/H4 diplotype. Interestingly, this negative trend of H4 in FHR-1 was less pronounced for FHR-1/2. Furthermore, the positive effect of H4 and negative effect of H2 on FHR-1 and FHR-2 levels, respectively, only significantly impacted FHR-1/2 levels when these haplotypes were combined. Diplotypes containing H2 showed a decrease in FHR-2/2, FHR-2, and FHR-4 levels, emphasizing H2’s pronounced negative effect on protein level. While the positive influence of H4 on FHR-2/2 and FHR-2 was visible in H1/H4 and H3/H4 diplotypes, this effect was mitigated by H2’s negative influence in H2/H4. Similar, the impact of H2 on FHR-5 levels was moderate and often neutralized by other haplotypes. Lastly, the higher levels of FHL-1 and FHR-3 in H3 were consistent across diplotypes. In contrast, where homozygous H3 and H4 showed either decreased or increased FH levels, respectively. In heterozygous diplotypes, these effects were less distinct, and FH levels were only decreased (H3) or increased (H4) when combined with H1.

The current concept is that the FHR proteins compete with FH and FHL-1 for binding to surface ligands on host cells, fine-tuning their function, though the exact role of the FHRs remains unclear [17, 18]. This knowledge gap is partly due to the limited insights into the physiological levels of these proteins and how they are influenced. Genetic analysis and measurement of several individual members have identified distinct haplotypes and associations with a wide range of diseases [41]. However, a full assessment of systemic levels of the entire family in a healthy population, while considering the known genetic heterogeneity is lacking. In this study, we report reference intervals for each member of the FH protein family in healthy blood donors. Additionally, we showed that common CFH haplotypes give rise to distinct protein expression levels across the whole family. First, to establish accurate baseline levels of the FH protein family in healthy donors, we investigated whether the timing of sample collection could impact their levels. We collected samples from eighteen donors over the course of 1 year and found consistently stable protein levels with minimal variation, demonstrating that the timing of sample collection within a relative short period does not significantly impact levels of the FH protein family in a healthy population.

When studying the complement system, serum is typically the preferred matrix for assessing complement activity, whereas EDTA plasma is favoured when quantifying complement activation products [42‒44]. To better understand the impact of sample matrix on the measurement of the FH family, we collected paired serum, along with three commonly used plasma types: EDTA, heparin, and citrate. Although statistically significant differences were observed for several FH family members when performing paired analysis, it is unclear if these differences are biologically relevant as the majority of donors fell within the range of assay variability. In general, using citrate plasma is not recommended as the sample is diluted upon collection and incorrect sample collection can result in further unwanted variation. Lastly, the use of heparin plasma was previously discouraged due to its interaction with several FH family proteins, i.e., FH, FHR-1, and FHR-5 [6, 7, 45]. In our study, we did not observe any significant differences in heparin plasma for FH and the FHRs. Interestingly, we did observe lower serum levels of FHL-1 compared to all three plasma types. Inducing clot formation in both citrate and EDTA plasma resulted in a decline of FHL-1 levels, suggesting FHL-1 may be consumed or degraded during coagulation [46]. The possibility of FHL-1 becoming encapsulated within the clot structure is unlikely, as smaller (FHR-2) and larger (e.g., FHR-4) members of the FH protein family did not exhibit a similar trend. It is well-established complement and coagulation are closely interconnected with previous studies demonstrating FH’s involvement in coagulation through interactions with factors such as FXIa, FXIIIa, and fibrin among others [47‒50]. Although the exact binding sites for these interactions are not yet fully understood and FHL-1 being highly similar with FH, FH levels itself were not affected by coagulation. However, a small decrease in FHL-1 may be more easily detected as it is less abundant in plasma compared to FH. Nevertheless, these data may suggest a yet unknown, but specific role of FHL-1 in clot formation and future research using inhibitors (e.g., hirudin and tridegin among others) specifically targeting proteins of coagulation is needed. In summary, our data show that when assessing the FH protein family in case/control studies, only samples with the same matrix, preferably EDTA plasma, should be directly compared.

To date, the utilization of unvalidated reagents, along with the use of diverse molecular techniques such as ELISA and targeted mass spectrometry among others, has resulted in a wide spectrum of reported levels and a lack of consensus regarding the physiological concentrations of the FH protein family, as reviewed by Poppelaars et al. [51]. Furthermore, specific quantification of each member of the FH protein family has proven challenging due to their high degree of amino acid similarity. This is particularly true for FHL-1 [14, 15]. Previously, reported concentrations of FHL-1 have varied, with values ranging from 0.04 to 1 µm, based on semiquantitative Western blot analysis or indirect ELISA methods [52, 53]. In this study, using a specific sandwich FHL-1 ELISA, we determined systemic serum levels of FHL-1 to be 0.023 µm (IQR = 0.018–0.028 µm). Additionally, considering that FHR-1, -2, and FHR-5 circulate in blood as homo- and heterodimers, it is difficult to accurately interpretate the in vivo situation when studies report total or inferred protein levels [54‒58]. This is particularly the case for studies employing targeted mass spectrometry as used in Zhang et al. [57, 59], Cipriani et al. [60] and Tierney et al. [58]. While mass spectrometry can provide target specificity based on mass and fragmentation, the inherent dynamic nature of FHR dimerization restricts mass spectrometry to quantify them. Previous studies, including our group, have demonstrated the impact of protein dimerization on ligand affinity [6, 7]. Furthermore, we recently showed that FHR-2 levels dictate the distribution of FHR-1 and FHR-2 dimers, highlighting the importance of quantifying individual dimer species rather than reporting only total protein levels [36]. In this report, we have provided systematic reference intervals for all proteins belonging to the FH protein family, encompassing all the distinct dimer species observed in vivo. These findings can be used to support the interpretation of circulatory levels in prospective and retrospective studies or used as baseline for designing more informative functional assessments of this protein family. Additionally, the reported low plasma concentration of FHL-1 and the FHRs compared to FH underscore the current concept that FHRs act more localized rather than systemically controlling the AP of complement.

Genetic variation within the CFH locus have been linked to a wide array of diseases [41]. However, most studies have focused on individual FH family proteins within the context of a specific disease. In this study, we demonstrated how the four predominant CFH haplotypes correspond to distinct systemic levels of each FH protein family member in a cohort of healthy Caucasian individuals. Our analysis highlights the significant reduction in FHR-2 and -4 within the H2 haplotype. The decreased FHR-2 levels in H2 were hypothesized earlier, with rs4085749 suggested as the causal SNP, a finding that has recently been confirmed at the protein level by our group and corroborated by others [21, 36, 60, 61]. Additionally, rs4085749 is in complete LD (D' = 1.0, r² = 1.0) with rs7531555, an established independent pQTL associated with lower CFHR4 levels, explaining the reduction of FHR-4 we observed in haplotype H2. Beyond rs7531555, two additional independent CFHR4 pQTLs with opposing effects were identified within H1: rs61818956 (increased), and rs10494745 (decreased). These findings underscore FHR-4 levels in haplotype H1 are substantially influenced by additional genetic markers, contributing to a wide variation in FHR-4 levels we observed within this common haplotype [21, 62].

Previous work by our group showed individuals that carry an FHR-3 polymorphism, frequently found in haplotype H3, associated with elevated levels of FHR-3 and a risk factor for aHUS [29]. Although our NGS panel did not cover the genetic regions tagging this CFHR3 polymorphism, its presence in haplotype H3 may explain the elevated FHR-3 levels within our cohort. Furthermore, the decreased FH levels we observed in H3 could potentially be attributed to the minor allele of the previously identified CFHR3 variant rs75703017. This is a SNP located in a liver-specific regulatory region and capable of modulating CFH expression at the genomic level [63]. Moreover, like the major allele of rs75703017, the common CFHR3/CFHR1 deletion, distinctive for haplotype H4, was found to associate with increased levels of FH, an association consistent with our findings [30].

Lastly and surprisingly, knowing CFH expression is modulated by variants in CFHR3, we have observed that levels of FHL-1 correlated with levels of FHR-3 across the common CFH haplotypes, whereas no correlation was observed between levels of FH and levels of FHL-1 or FHR-3 [30]. Interestingly, this lack of correlation highlights the cell-specific regulation of alternative splicing of FH which appears to associate with the expression of FHR-3 [64, 65]. Although these common haplotypes result in distinct systemic concentrations across the entire FH protein family, further research is required to investigate how, in addition to known qualitative genetic mutations in CFH, quantitative genetic variations in CFHR contribute to an individual’s complotype and confers either risk or protection in complement-associated diseases [38]. These findings further underscore the intricate and interconnected nature of the FH protein family at both the genomic and protein level, emphasizing the complexity of their regulation and potential functional interactions in health and disease.

This study has several limitations. First, the size of the cohort was too small to capture the full range of CFH haplotypes, excluding rare CFH sub-haplotypes (H5–H7) or low frequency SNPs at the CFH locus [26, 61]. While we adopted the most accepted method for haplotyping the CFH locus, this approach may overlook alternative haplotyping methods. Additionally, our study was limited to exon sequencing of the CFH locus, restricting our ability to accurately pinpoint previous identified causal quantitative intronic SNPs. Moreover, this study was conducted within a population of Western ancestry, thereby excluding genetic variations known to exist in other ethnic groups. For instance, the frequency of homozygous CFHR3/CFHR1 deficiency varies globally, with a high prevalence in populations of African ancestry (3.4–33.3%), while the deletion is extremely rare in Asian populations [33, 66, 67]. Furthermore, the study included healthy adults aged 22–64 years, excluding children and elderly people. Finally, the current study focuses on the circulatory levels of the FH protein family, emphasizing the need for measurements in matrices beyond serum or plasma such as urine, bronchial-, spinal-, or synovial fluid, among others. Having reference intervals in such matrices is particularly relevant in the context of complement-associated diseases, where specific organs like the kidney, joint or the eye may be affected [68].

In summary, by using ELISAs, we reported systemic reference levels of each member of the FH protein family found in vivo. Furthermore, we demonstrated how common haplotypes of the CFH locus led to distinct expression patterns across the entire family. The established reference intervals and identified genetic effects provide a foundation for further research and emphasizes the importance of including all family members when studying their role in both health and disease.

The authors would like to thank the healthy volunteers for participating in this study. In addition, we would like to thank Maaike Derlagen and Nadia C.H. Keijzer from Sanquin Diagnostic Services for their assistance in sequencing the healthy donors.

After consultation with the Medical Ethical Committee of Sanquin Research, Amsterdam, The Netherlands, a system was established for obtaining blood samples for scientific research (no Approval No. available). This volunteer system is organized according to Dutch regulations and according to the Declaration of Helsinki. This volunteer system certifies, among others, that: blood samples used for scientific studies by researchers of the Sanquin Research department were drawn from healthy, anonymized volunteers with written informed consent; no personal characteristics of the volunteers are registered; niether the volunteers nor those taking the samples know for what project specific samples are used; allowed annual sample volume and frequency of donation were established after consultation with Sanquin Medical Secretary. Standard operating procedures are available upon request.

E.J.M.T. and M.R. are employees of Hycult Biotech. R.P., M.C.B., and T.W.K. are coinventors on patents and patent applications describing the therapeutic use of anti-FH antibodies. All other authors declare no conflict of interest. G.S.H. is a shareholder, consultant, and cofounder of Perceive Biotherapeutics, LLC. G.S.H., and B.T.R. are inventors on patents and patent applications owned by the University of Utah.

This research was supported by the European Union’s Horizon 2020 Research and Innovation Program under grant agreement No. 899163 (SciFiMed) and by the Sanquin Research Fund Young Investigator Award to B.R.J.V. The funders had no role in the design, data collection, data analysis, and reporting of this study.

B.R.J.V., M.R., E.J.M.T., T.W.K., and R.B.P. designed the research. B.R.J.V., E.C., M.R., M.C.B., and G.M. conducted experiments and gathered data. J.G., K.L., MD, and NCHK genotyped healthy donors and analysed the data. B.T.R., J.L., R.A.A., and G.S.H. contributed to FHL-1 determination. B.R.J.V. and R.B.P. wrote the first draft of the manuscript. M.R. and E.J.M.T. critically evaluated the manuscript. All authors contributed to the article and approved the final version for publication. They agreed to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Bert R.J. Veuskens and Mara van Rossum contributed equally and share first authorship. Erik J.M. Toonen and Richard B. Pouw contributed equally and share last authorship.

All data supporting the results in this article are included in this article and its supplementary material files. Further queries can be directed to the corresponding author (R.B.P., [email protected]).