Complete functional characterization of disease-associated genetic variants in the complement factor H gene

clinical investigation

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Complete functional characterization of disease-associated genetic variants in the complement factor H gene He´ctor Martı´n Merinero1, Sheila Pinto Garcı´a1, Jesu´s Garcı´a-Ferna´ndez1, Emilia Arjona1, Agustı´n Tortajada2 and Santiago Rodrı´guez de Co´rdoba1 1

Centro de Investigaciones Biológicas and Ciber de Enfermedades Raras, Madrid, Spain; and 2Department of Immunology, Complutense University School of Medicine and 12 de Octubre Health Research Institute (imas12), Madrid, Spain

Genetic analyses in atypical hemolytic uremic syndrome (aHUS) and C3-glomerulopathy (C3G) patients have provided an excellent understanding of the genetic component of the disease and informed genotypephenotype correlations supporting an individualized approach to patient management and treatment. In this context, a correct categorization of the disease-associated gene variants is critical to avoid detrimental consequences for patients and their relatives. Here we describe a comprehensive procedure to measure levels and functional activity of complement regulator factor H (FH) encoded by CFH, the commonest genetic factor associated with aHUS and C3G, and present the results of the analysis of 28 uncharacterized, disease-associated, FH variants. Sixteen variants were not expressed in plasma and eight had significantly reduced functional activities that impact on complement regulation. In total, 24 of 28 CFH variants were unambiguously categorized as pathogenic and the nature of the pathogenicity fully documented for each. The data also reinforce the genotype-phenotype correlations that associate specific FH functional alterations with either aHUS or C3G and illustrate important drawbacks of the prediction algorithms dealing with variants located in FH functional regions. We also report that the novel aHUS-associated M823T variant is functionally impaired. This was unexpected and uncovered the important contribution of regions outside the N-terminal and C-terminal functional domains to FH regulatory activities on surfaces. Thus, our work significantly advances knowledge towards a complete functional understanding of the CFH genetic variability and highlights the importance of functional analysis of the disease-associated CFH variants. Kidney International (2018) 93, 470–481; http://dx.doi.org/10.1016/ j.kint.2017.07.015 KEYWORDS: C3 glomerulopathy; complement; hemolytic uremic syndrome Copyright ª 2017, International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Correspondence: Santiago Rodríguez de Córdoba, Centro de Investigaciones Biológicas, Ramiro de Maeztu 9, 28040 Madrid, Spain. E-mail: [email protected] Received 22 June 2017; revised 18 July 2017; accepted 20 July 2017; published online 21 September 2017

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actor H (FH) is the key regulator of the alternative pathway (AP) of the complement system. FH controls complement activation, both in the fluid phase and on cellular surfaces, preserving complement homeostasis and preventing uncontrolled C3b deposition and host tissue damage. The FH regulatory activities include binding to C3b, blocking C3b-factor B interaction during the assembly of the AP C3-proconvertase complex (C3bB); acceleration of the decay of the AP C3-convertase (C3bBb); and acting as cofactor of factor I (FI) in the proteolytic inactivation of C3b.1 FH is a relatively abundant plasma protein that is secreted as a single-chain glycoprotein of 155 kDa composed of 20 homologous domains of w60 amino acids, called short consensus repeats (SCRs). FH concentration in plasma is highly variable, ranging from 76 to 247 mg/ml. Along the 20 SCR of FH, there are different interaction sites for C3b and polyanions. The SCR1-4 region is the only C3b binding site acting as a cofactor for FI to cleave C3b and to accelerate the decay of AP C3-convertase. Similarly, the C3b and polyanion binding site at SCR19-20 determines the ability of FH to bind surface-bound C3b, this region of FH being essential for self-pathogen discrimination.2,3 In addition to these regions, it was recently postulated that a central FH segment, including SCR14, facilitates FH bending and increases avidity for C3b by enabling the simultaneous binding of FH to different sites in C3b.4,5 Pathogenic variants and polymorphisms in the CFH gene are associated with a number of diseases, including atypical hemolytic uremic syndrome (aHUS) and C3-glomerulopathy (C3G).6 aHUS is a thrombotic microangiopathy characterized by thrombocytopenia, hemolytic anemia, and acute renal failure. The primary pathogenic event in aHUS is endothelial cell injury caused by complement dysregulation.7 C3G is a very rare form of glomerulonephritis, characterized by the presence of electron-dense deposits within the glomerular basement membrane.8 C3G is associated with complement abnormalities that lead to a persistent reduction of C3 serum levels and to intense deposition of degradation products of C3 in the glomerular basement membrane.9,10 Over the past 15 years, genetic analyses in aHUS and C3G patients have shown that variants in CFH are the most common genetic alteration associated with these disorders. However, we have a limited understanding of the functional Kidney International (2018) 93, 470–481

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HM Merinero et al.: Complete functional analysis of CFH variants

consequences of almost one-third of the genetic variants identified in CFH, which is a potential cause of misinterpretations with important consequences for the patients and their relatives. We report here the development of a comprehensive analytical procedure for the functional characterization of FH and demonstrate its effectiveness in identifying the expression and functional consequences of a large number of novel aHUS and C3G-associated CFH variants. The data allowed us to categorize them as pathogenic or benign and reinforced our current understanding of the pathogenic mechanisms underlying aHUS and C3G. RESULTS Novel FH variants found in the genetic analysis of aHUS and C3G patients

A total of 707 aHUS and 234 C3G patients included in the Spanish aHUS/C3G Registry (https://www.aHUSC3G.es) were screened for CFH variants, which resulted in the identification of 101 CFH variants, 87 in aHUS and 35 in C3G; 21 variants were associated with both pathologies. As a whole, 123 aHUS patients (17.4%) and 27 C3G patients (11.5%) carried CFH gene variants. Notably, 28 of these CFH variants (27.5%) were novel variants for which the functional consequences were unknown (Table 1). In addition, these patients were screened for all aHUS and C3G candidate genes. A summary of the clinical and genetic characteristics of the patients carrying these novel CFH variants is presented in Table 2 to illustrate that most patients presented with severe disease phenotypes. All other CFH variants (N ¼ 73) found in our patients were previously described, and their functional consequences are known.11 We named those variants located in SCR1-4 (R53C, R53P, R53S, S58A, C66Y, L98F, A110S, Y118Ifs*4, C192W, and Y235C) N-terminal, those in SCR1920 (D1119N, P1166L, R1182K, C1218R, and c.3493þ1G>A) were named C-terminal, and variants located in SCR5-18 (C309W, C320R, K331E, C597*, K670T, M823T, R830W, C853R, C853Y, R885Sfs*13, T956M, W1037*, and c.29571G>A) were named central region. A number of asymptomatic relatives carrying the CFH gene variants were also identified (Table 1). A search for these CFH variants in the Spanish and European control populations illustrated, that with only 3 exceptions (S58A, R830W, and T956M), all other CFH variants have not been previously encountered (Supplementary Table S1). To predict the functional consequences of the 28 novel CFH variants detected in this study, we used several prediction algorithms available in ANNOVAR software. We considered likely pathogenic those variants in which a deleterious impact of the genetic variation was anticipated by a majority of these prediction algorithms. According to these analyses, the CFH variants R53C, R53S, C66Y, C192W, Y235C, C309W, C320R, C853R, C853Y, D1119N, and C1218R were considered likely pathogenic, whereas R53P, S58A, L98F, A110S, K331E, K670T, M823T, R830W, T956M, P1166L, and R1182K were predicted to be benign or variants of uncertain significance (Supplementary Table S2). For the C597*, W1037*, Y118Ifs*4, Kidney International (2018) 93, 470–481

and R885Sfs*13 variants, some of the prediction algorithms failed to give a result. However, the nature of these CFH variants, introducing a stop codon, justifies their assignment to the category of likely pathogenic variants. Similarly, c.2957-1G>A and c.3493þ1G>A were predicted to be pathogenic because they alter the splicing sequences. As a whole, the analyses to predict pathogenicity indicate that 11 of the 28 CFH variations (39%) are predicted to be either benign or variants of uncertain significance (Supplementary Table S2). To formally categorize the diseaseassociated CFH variants and to establish their causal relationship with the pathology, we determined their expression levels in plasma and purified those that were expressed to perform a complete functional characterization. To do this, we benefited from having serum or plasma samples from all patients and relatives and the Y402H genotypes available for all individuals (Table 1). FH plasma levels

Twenty-one of the 28 CFH variants are carried by Y402H heterozygote individuals (patients and/or relatives). In all of these cases, a direct measurement of the FH produced by the mutated CFH allele was obtained. These analyses allowed us to conclude that the C66Y, Y118Ifs*4, C192W, Y235C, C309W, C320R, C597*, C853R, C853Y, R885Sfs*13, W1037*, and C1218R CFH variants are not expressed or result in very low levels of FH in plasma. We also concluded that the L98F, c.2957-1G>A, and c.3493þ1G>A variants are not expressed because total plasma levels in heterozygote carriers were <50% of the normal FH plasma levels (Supplementary Figure S1). In addition, we found that the A110S CFH variant, albeit present in plasma, it is significantly reduced compared with normal FH levels (Table 1 and Supplementary Figure S1). The normal range of variation for FH plasma levels produced by a single allele and the normal range for total FH plasma levels in control individuals were previously determined to be 50 to 127 mg/ml and 76 to 247 mg/ml, respectively (Supplementary Figure S1). To functionally characterize the CFH variants that are expressed (R53C, R53S, S58A, K331E, K670T, T956M, and P1166L), we purified them to homogeneity from the plasma or serum of Y402H heterozygote carriers (Figure 1b). The expressed CFH variants for which no Y402H heterozygote carriers were available (R53P, M823T, R830W, D1119N, and R1182K) were purified together with the wild-type FH protein (Figure 1c). Alternative pathway regulatory activity of the disease-associated FH variants

Six of the 12 purified FH variants (R53S, S58A, M823T, D1119N, P1166L, and R1182K) present significant reduced capacity to regulate the activity of the alternative pathway in our modified sheep red blood cell (SRBC) hemolytic assay (Figure 2). Notably, K331E showed significantly reduced lysis, which suggests improved surface protective capacity. It should also be mentioned that among the 5 variants that showed no 471

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HM Merinero et al.: Complete functional analysis of CFH variants

Table 1 | Novel uncharacterized CFH variants associated with aHUS and C3G in Spanish patients Variant c.157C>T c.157C>A

c.158G>C

c.172T>G

Protein

Exon (SCR)

R53C R53S

2 (1) 2 (1)

R53P

S58A

2 (1)

2 (1)

c.197G>A

C66Y

2 (1)

c.292C>T c.328G>T

L98F A110S

3 (2) 3 (2)

c.350delG

Y118Ifs*4

3 (2)

c.576T>G c.704A>G

C192W Y235C

5 (3) 6 (4)

c.927C>G

C309W

7 (5)

c.958T>C

C320R

7 (5)

c.991A>G

K331E

8 (6)

c.1791C>A c.2009A>C c.2468T>C

C597* K670T M823T

13 (10) 14 (11) 17 (14)

c.991A>G

R830W

17 (14)

c.2557T>C

C853R

17 (14)

c.2558G>A

C853Y

17 (14)

c.2655del

c.2940C>T c.2957-1G>A

R885Sfs*13

18 (15)

T956M

19 (16)

-

Intron 19

c.3110G>A

W1037*

20 (17)

c.3355G>A

D1119N

22 (19)

c.3493þ1G>A c.3497C>T

— P1166L

Intron 22 23 (20)

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Plasma levels (mg/ml)

Carrier ID

Status

Tyr402His genotypes

N-terminal region 1 2 2-M 2-P1 3 3-M 3-P1 4 4-P4 4-P8 5 6 6-P 7 8 8-P1 8-P2 8-P3 8-P4 8-P5 8-P6 8-P7 8-P8 9 9-P1 10 11 12

aHUS aHUS Healthy Healthy aHUS Healthy Healthy aHUS Healthy Healthy aHUS aHUS Healthy aHUS C3G Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy C3G Healthy aHUS C3G aHUS

Ha/Ha Ya/Y Ya/H Ya/Y Ya/Y Ya/Y Ya/H Ha/H (null)b Y/Ha Y/Ha Y/Ha Ya/H Ya/Y Ya/Y Ha/Ha Y/Ha Y/Ha Y/Ha Y/Ha Ha/Ha Ha/Ha Y/Ha Y/Ha Ya/H Ya/Y Ya/Y Ya/H Y/Ha

Central region 13 13-P2 13-P4 14 14-P4 15 15-P 16 17 18 18-M 19 19-P 20 20-P2 20-P3 21 21-P4 22 22-P1 23 24 24-M 25 25-P1 25-P7 26

aHUS Healthy Healthy aHUS Healthy C3GN Healthy aHUS aHUS aHUS Healthy C3G Healthy aHUS Healthy Healthy aHUS Healthy aHUS Healthy C3G aHUS Healthy aHUS Healthy Healthy aHUS

Y/Ya Ya/H Y/Ya Ya/Y Ya/H Y/Ha H/Ha Y/Ha Y/Ha Ya/Y Ya/Y Ya/Y Ya/Y Y/Ha Y/Ha Y/Ha Ya/Y Ya/H Y/Ha H/Ha Ha/Ha H/Ha Y/Ha Ya/Y Ya/Y Ya/Y Y/Ha

75 0 89 58 0 108

C-terminal region 27 27-P 28 29 30

aHUS Healthy aHUS aHUS aHUS

Ya/Y Ya/Y Ya/Y Ya/Y Ya/H

178 91 43 139 60

Tyr402

His402

c

172

242 84 138 168 206 144 c

112 128 96 0 47 75 c

114 74 68 94 c c

75 88 0 62 58 0 128

c

51 90 161 166 149 161 113 100 82 52 0 93 c c c

59 75 79 72 80

c

98 c c c

81 51 57 58 62 64 c c

38 18 23 12 17 33 28 18 16 78 c c

65 0 c

99 c c

89 90 145 0 102 c c c c

0 0 0 c

101 0 62 0 241 79 c c c

0 c c c c

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HM Merinero et al.: Complete functional analysis of CFH variants

Table 1 | (Continued) Variant

Protein

c.3545G>A

Exon (SCR)

R1182K

c.3652T>C

23 (20)

C1218R

23 (20)

Carrier ID

Tyr402His genotypes

Status

30-M 31 31-M 31-P1 31-P2 31-P3 32

Plasma levels (mg/ml) Tyr402

His402

90 183 172 164 96 67 60

103

a

Healthy aHUS Healthy Healthy Healthy Healthy aHUS/C3G

Y /H Ya/Y Ya/Y Ya/Y Ya/H Ya/H Y/Ha

c c c

92 69 0

a

Allele carrying the disease-associated variant. Compound heterozygote with a null CFH allele. Indicates the absence of an allele because the individual is homozygous for the other allele.

b c

significant differences with the wild-type FH protein, 2 of them (R53P and R830W) were copurified together with the FH wild-type allele. Notably, all the variants associated with

the loss of regulatory capacity are located in functional regions (SCR1-4 and SCR19-20), except M823T, which is located in SCR14 within the central region of FH.

Table 2 | Clinical and genetic data of patients carrying CFH variants Variant

Age, yr

Sex

Onset age (yr)

Outcome

Transplant

Recurrences

Treatment

aHUS 1 2 3 4

R53C R53S R53P S58A

24 40 38 58

F F F F

2 36 35 47

CKD CR N/A CKD

0 0 0 N/A

No Yes N/A No

Eculizumab Plasma treatment N/A N/A

5 6 7

S58A C66Y L98F

6 36 36

M F M

3 12 4

CR CKD CKD

0 3 2

N/A Yes Yes

N/A Eculizumab Eculizumab

10 12 13 14 16 17 18

Y118Ifs*4 Y235C C309W C320R C597* K670T M823T

61 52 51 3 16 8 10

F M F F F F F

42 34 48 1 13 4 1

CR CKD N/A CR CR CR CR

0 2 N/A 0 0 0 0

Yes Yes N/A No N/A No Yes

Plasma treatment N/A Eculizumab Eculizumab Eculizumab N/A Eculizumab

20 21 22

C853R C853Y R885Sfs*13

67 49 62

M M M

53 41 59

CKD CKD N/A

1 0 0

N/A N/A N/A

Plasma treatment N/A Eculizumab

24 25 26

T956M c.2957-1G>A W1037*

30 50 49

M M F

1 46 47

N/A CR CKD

N/A 0 0

N/A Yes N/A

Plasma treatment N/A Eculizumab

27 28 29

D1119N c.3493þ1G>A P1166L

9 36 56

F F M

1 30 45

N/A N/A CKD

0 N/A 1

N/A N/A N/A

N/A N/A N/A

P1166L R1182K C1218R

38 50 39

F F F

29 36 29

CKD CKD CR

1 0 0

N/A N/A Yes

Eculizumab N/A Plasma treatment

A110S

64

M

57

N/A

1

Yes

Y118Ifs*4 C192W K331E R830W R885Sfs*13

60 45 10 14 53

M M M F F

25 1 NA NA 17

CKD N/A N/A N/A CKD

0 0 N/A N/A 2

N/A Yes N/A N/A Yes

Eculizumab, prednisone N/A Prednisone Prednisone N/A Prednisone

Carrier ID

30 31 32 C3G 8 9 11 15 19 23

Additional genetic risk factors No No No CFH: c.88dup (p.T30Nfs*10) No No MCP: c.800_820del (p.T267_N273del) No No No No No No CFI: c.1149-2 A>G; c.1643A>G (p.Q548G) No No C3: c.193A>C (p.K65Q) No MCP; c.286þ1G>C THBD: c.1502C>T (p.P501L) No No CFI: c.1322A>G (p.K441R) No No No No No No No No C3: c.193A>C (p.K65Q)

aHUS, atypical hemolytic uremic syndrome; CKD, chronic kidney disease; CR, complete remission; F, female; M, male; N/A, data no available. Kidney International (2018) 93, 470–481

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HM Merinero et al.: Complete functional analysis of CFH variants

Figure 1 | Purification of FH variants. (a) A flow chart of the purification protocol of the FH variants. (b) Coomassie-stained gels of FH variants purified to homogeneity from Y402H heterozygotes and (c) FH variants that were purified together with the FH wild-type allele because the carrier was a Y402 or H402 homozygote. EDTA, ethylenediamine tetraacetic acid; FH, factor H; mAb, monoclonal antibody.

Characterization of specific functional alterations in the CFH variants

We next analyzed the functional impairment of the CFH variants that are expressed in plasma. Decay-accelerating activity (DAA) on surfaces was analyzed by surface plasmon resonance (Figure 3). Our results revealed that R53C and R53S completely lack DAA, whereas R53P, S58A, M823T, D1119N, P1166L, and R1182K show significantly reduced DAA compared with the wild-type FH protein (Figure 3). The FI-dependent cofactor activity of the proteolytic inactivation of C3b was tested in the fluid phase using purified proteins. The results indicate that R53C, R53S, S58A, and M823T present significantly reduced fluid-phase FI cofactor activities compared with wild-type FH protein (Figure 4). Finally, we analyzed the capacity to bind C3b by the C-terminal region. In these analyses, the FH variants were captured to the surface plasmon resonance chip by the N-terminal region so that they can only bind C3b by the C-terminal region (see Materials and Methods). Altogether, these analyses revealed that only the FH variants D1119N, P1166L, and R1182K present significantly decreased C-terminal C3b binding compared with the wild-type FH protein (Figure 5). As a whole, these analyses show that 9 of the 12 expressed CFH variants show impaired complement regulatory activities. Adding these 474

variants to those that are not expressed allowed us to unambiguously categorize 24 of the 28 CFH variants as pathogenic (Table 3). Importantly, although all CFH variants that were predicted in silico to be pathogenic variants were experimentally confirmed to be pathogenic, as many as 63% (7/11) of the CFH variants that were predicted in silico to be benign were shown experimentally to be pathogenic (Table 3).

DISCUSSION

This study provides a complete analysis of 28 uncharacterized CFH variants found in 32 patients with a diagnosis of aHUS and C3G (Table 2). This was achieved by developing a comprehensive analytical procedure that allowed us to determine allele-specific plasma levels of the FH variant and to purify and perform a full functional characterization of the variants that are expressed in plasma. A flow chart of the analytical procedure is depicted in Figure 6. The 28 CFH variants included in these analyses were scattered along the whole FH amino-acid sequence and involved known functional FH domains as well as nonfunctional FH regions (Table 1). Sixteen CFH variants were associated with decreased plasma levels of FH, 12 of which Kidney International (2018) 93, 470–481

HM Merinero et al.: Complete functional analysis of CFH variants

clinical investigation

Figure 2 | Overall functional activity of the FH variants. The individual SRBC hemolytic analysis for each of the expressed FH variants that could be purified from the plasma of appropriate carriers. FH variants are indicated by open circles. Wild-type FH is indicated by solid circles. Maximum NHSDFH-induced lysis is indicated as 100%. EC50 (50% effective concentration) values for each FH variant indicating the relative amounts required to achieve the same protection as wild-type FH. P values for each comparison are indicated. FH, factor H.

were unambiguously explained by the lack of expression of the mutated CFH allele (Table 1 and Supplementary Figure S1). Because no Y402H heterozygote samples were available to test FH allele–specific expression of the L98F, c.2957-1G>A, and c.3493þ1G>A CFH variants, lack of expression of these alleles was postulated based on the >50% decreased total FH plasma levels observed in carriers of these variants (Table 1 and Supplementary Figure S1). The deleterious consequences of these CFH variants, introducing significant changes in the amino acid sequence of FH or altering the splice regions, were anticipated by the prediction algorithms, with only 1 exception (Table 3). The exception is L98F, the expression of which was clearly found decreased in our allele-specific enzyme-linked immunosorbent assay (Table 1 and Supplementary Figure S1), but the variant was predicted to be benign in silico. However, we cannot exclude that this low expression is a consequence of another sequence variation in the regulatory regions of this particular CFH gene variant that were not included in our DNA sequencing analysis. All expressed CFH variants were purified from plasma for further analyses. These include the 4 N-terminal variants R53C, R53P, R53S, and S58A. Impairment of the FH complement regulatory activity using our modified SRBC Kidney International (2018) 93, 470–481

hemolytic assay could be demonstrated only for R53S and S58A, but DAA was absent or significantly reduced in all of them, and the FI-cofactor activity was only normal in R53P, although this last finding may be related to the fact that the R53P CFH variant was copurified with the wild-type allele. These and previous data obtained with the recombinant FH mutants R53C12 and R53H,13 demonstrating that these 2 variants have impaired DAA and FI-cofactor activity, illustrate that amino acid R53 is a critical residue for the FH regulatory activities. Three of the 5 CFH variants located in the C-terminal SCR19-20 (D1119N, P1166L, and R1182K), were also expressed and showed reduce surface protection in the SRBC hemolytic assay, reduced DAA, and impaired C-terminal C3b binding. M823T in SCR14 is the only CFH variant located outside the N-terminal and C-terminal functional regions that is expressed and that revealed a functional alteration. It showed increased lysis in the SRBC hemolytic assay and reduced DAA and FI-cofactor activity. Notably, the C3b binding activity mediated by the C-terminal region was normal (Figure 5). This suggests that M823 residue may be implicated in the bending of the FH protein, allowing the simultaneous interaction of the N-terminal and C-terminal C3b binding sites with the same 475

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HM Merinero et al.: Complete functional analysis of CFH variants

Figure 3 | SPR analysis of the decay-accelerating activity of the FH variants. The alternative pathway C3 convertase was assembled by flowing over a C3b-coated chip during 150s factor B (400 nM) and factor D (20 nM). (a) The gray rectangle illustrates the spontaneous decay (dashed line) and the FH-mediated decay-accelerating activity (DAA) (solid line). (b) Fragments of the surface plasmon resonance (SPR) sensograms to illustrate the DAA of N-terminal variants R53C, R53P, R53S, and S58A; the central region variant M823T; and C-terminal variants D1119N, P1166L, and R1182K, respectively. P values for the difference in the wild-type FH are indicated: *P < 0.05, **P < 0.001.

C3b molecule.4,14 Notably, it has been proposed that the region including SCR14 organizes a zigzag structure that permits the FH molecule to bend back, approaching the N- and C-terminal regions and allowing them to interact with distinct sites in the C3b molecule, which increases avidity and regulatory efficacy. This model postulates the existence of side-by-side interaction between SCR12-13 and SCR14-15, interactions in which M823T could be implicated (Figure 7). The contribution of these central region FH domains to the overall regulatory activity of FH is further supported by the observation that FHL-1 has lower FI-cofactor activity compared with FH,15 supporting that the absence of the C-terminal C3b binding domain results in reduced avidity of FHL-1 for C3b. Our interpretation of the observed alteration of both DAA and FI-cofactor activity is, therefore, that the M823T variant causes a reduced capacity to bend back or impairs the stability of the bent structure. Experiments, beyond the scope of this report, are in progress to determine the precise contribution of SCR14 to the overall structure of FH. Our results confirm that in silico prediction of pathogenicity in functionally relevant regions of the FH molecule is unreliable using the current algorithms because it fails to assign a correct categorization in almost two-thirds of the CFH variants. This drawback should be expected because 476

these algorithms are designed mainly to identify molecular pathogenicity (how well the protein architecture tolerates the genetic change) and not how the sequence variations alter the specific functional activities of the protein. Consistent with this, we observed that 100% of the CFH variants predicted in silico to be pathogenic were indeed demonstrated experimentally to be pathogenic, mainly because they were not expressed at the protein level. As a whole, these data illustrate that functional analysis is particularly important for novel CFH variants that have been categorized in silico as benign and are located in functionally relevant FH regions. Our data also provide further support of the genotypephenotype correlations that associate particular CFH mutations with aHUS or C3G.16 These genotype-phenotype correlations explain why the CFH gene is associated with both aHUS and C3G. In aHUS, the most prevalent CFH genetic alterations are missense mutations that alter the C3b- and polyanion-binding site at the C-terminus of FH. These aHUS-associated CFH variants rarely result in decreased FH plasma levels; they present normal regulatory activity in plasma but a limited capacity to protect cells from complement lysis.7 In contrast to aHUS, the prototypical C3G-associated CFH mutations result in FH deficiencies or amino acid substitutions that eliminate the complement Kidney International (2018) 93, 470–481

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clinical investigation

Figure 4 | Analysis of the factor I (FI)–dependent C3b cofactor activity of the factor H (FH) variants. The FI cofactor activity of the CFH genetic variants was assayed in vitro using purified proteins. Each graph corresponds to 1 CFH variant and represents a time course experiment showing the percentage of C3b cleaved, as determined by the a0 /b chain ratio. Data are the mean  SD of duplicates. Time required to cleave 50% of the C3b (50% effective time [ET50]) for the wild-type FH was 3.56  0.67 minutes. The ET50 for the FH variants are indicated as the times of ET50 for the wild-type FH with their corresponding P value. DAA, decay-accelerating activity; FB, factor B, FD, factor D; Resp. Diff., RU, resonance unit; WT, wild type.

regulatory activities located at the N-terminus of FH. These C3G-associated CFH mutations lead to unrestricted activation of complement in plasma, causing damage to glomerular

Figure 5 | Analysis of C3b binding by the C-terminal FH region. Two different amounts of each purified FH variant were captured to a surface plasmon resonance chip coated with OX24, a mouse monoclonal antibody that recognizes the N-terminal region of FH, and C3b was flown through the chip. Differences in the capacity to bind C3b between the FH variants and the wild-type FH protein were determined by comparing the slopes of the linear representations that result from plotting the RUs of captured FH versus the RUs of C3b bound to the captured FH. Kidney International (2018) 93, 470–481

cells and deposition of complement products in the glomerular basement membrane gene.9,10 In agreement with these data, the 5 CFH pathogenic variants found to be associated with C3G impair FH expression. One patient carries R885Sfs*13 in homozygosis, which resulted in complete FH deficiency, whereas the other 4 C3G patients carry partial FH deficiencies due to A110S, Y118Ifs*4, C192W, and C1218R CFH variants (Table 1). CFH variants associated with aHUS include both variants that are expressed normally but present impairment of complement regulatory activity on surfaces (R53C, R53P, R53S, S58A, M823T, D1119N, P1166L, and R1182K) and variants that cause partial FH deficiencies (L98F, Y118Ifs*4, Y235C, C309W, C320R, C597*, C853T, R885Sfs*13, c.2957-1G>A, W1037*, c.3493þ1G>A, and C1218R). Interestingly, 3 of these latter CFH variants (Y118Ifs*4, R885Sfs*13, and C1218R) are also associated with C3G, which again agrees with previous data indicating that a partial deficiency of FH predisposes to different disorders and that, in these cases, the disease phenotype is determined by additional genetic or environmental risk factors.16 In conclusion, we developed an analytical strategy for the complete characterization of novel CFH variants identified in the genetic studies of patients with pathologies associated 477

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Table 3 | Summary of functional analyses and comparison with the in silico data Variant R53C R53S R53P S58A C66Y L98F A110S Y118Ifs*4 C192W Y235C

Expression Yes Yes Yes Yes No No Very low No No No

Hemolytic assay Normal Affected Normal Affected

Decay accelerating activity

FI cofactor

C-ter C3b-binding

N-terminal region Affected Affected Affected Affected Affected Normal Affected Affected

Summary

ANNOVAR prediction

Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic

Pathogenic Pathogenic VUS Benign Pathogenic Benign VUS Pathogenic Pathogenic Pathogenic

Pathogenic Pathogenic Benign Pathogenic Benign Pathogenic Benign Pathogenic Pathogenic Pathogenic Benign Pathogenic Pathogenic

Pathogenic Pathogenic Benign Pathogenic VUS Benign VUS Pathogenic Pathogenic Pathogenic Benign Pathogenic Pathogenic

Pathogenic Pathogenic Pathogenic Pathogenic Pathogenic

Pathogenic Pathogenic VUS Benign Pathogenic

Central region C309W C320R K331E C597* K670T M823T R830W C853R C853Y R885Sfs*13 T956M c.2957-1G>A W1037*

No No Yes No Yes Yes Yes No No No Yes No No

D1119N c.3493þ1G>A P1166L R1182K C1218R

Yes No Yes Yes No

Normal

Normal

Normal

Normal Affected Normal

Affected

Affected

Normal

Normal

Affected

C-terminal region Affected

Affected

Affected Affected

Affected Affected

Affected Affected

VUS, variants of uncertain significance.

with complement dysregulation. We demonstrated its usefulness analyzing 28 novel CFH variants associated with aHUS and C3G and unambiguously categorizing 24 of them as pathogenic. Our data highlight the importance of the experimental data to provide a correct categorization of the diseaseassociated variants and to unravel potential novel functional regions in FH. In this respect, the functional implications of the M823T CFH variant warrant further investigations. As a whole, this work represents a significant step toward a complete functional understanding of the CFH genetic variability. MATERIALS AND METHODS Mutation screening and genotyping Patients and healthy relatives were screened for mutations in all aHUS and C3G candidate genes using different DNA-sequencing approaches (Table 1). Traditional DNA Sanger sequencing was used to screen early patients. More recently, we used next-generation sequencing. All gene variants found in the next-generation sequencing analyses were confirmed by Sanger sequencing. A summary of the clinical and genetic characteristics of the carriers of these genetic variants is provided in Table 2. Measurement of FH plasma levels. Total FH plasma levels and levels of FH determined by each CFH allele were measured from plasma samples by enzyme-linked immunosorbent assay using 2 different monoclonal antibodies (mAbs) that recognize specifically 478

the Tyr402 (MBI-6) or the His402 (MBI-7) variants of the common CFH polymorphism Y402H.17 Briefly, 96-well plates were coated with the rabbit polyclonal antibody anti-FH 34þ35 (in house) in phosphate-buffered saline, pH 7.4, overnight at 4  C. After blocking the plates with Tris-Tween (50 mM Tris, pH 7.4; 150 mM NaCl, 0.2% Tween 20 [Darmstadt, Germany]) containing 1% bovine serum albumin for 1 hour at room temperature, appropriate dilutions of the plasma samples in blocking buffer were added and incubated for 1 hour at room temperature. The FH Tyr402 and His402 variants were detected using the mAbs MBI-6 and MBI-7, respectively, followed by a peroxidase-labeled goat anti-mouse Igs antibody (DAKO, Glostrup, Denmark). For detection, we used ophenylenediamine dihydrochloride substrate and 10% sulfuric acid to stop the reaction. Absorbance was measured at 492 nm. A Y402H heterozygous plasma with known concentration of both variants was used as the standard curve. All samples were tested in duplicate. Protein purifications FH proteins were isolated from 3- to 5-ml plasma samples of FH Y402H from heterozygous patients or relatives.18 We combined 2 immuneaffinity chromatography columns, a CNBr-activated sepharose 4B (GE Healthcare, Sugar Notch, PA) column coupled with the in-house mAb anti-FH 214 and another coupled with the mAb MBI-7 (Figure 1a). Fractions containing FH were collected, concentrated (Amicon Ultra, 4–30 kDa, Millipore, Temecula, CA) and applied to a molecular exclusion column (Superdex200 Increase 10/300 GL, GE Kidney International (2018) 93, 470–481

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Identification of a novel genetic variant Affinity chromatography FH purification Search for a Y402H heterozygote carrier

Normal plasma levels

Hemolytic assay Y402H allele-specific ELISA Total FH ELISA

DAA

FI cofactor activity

C3b binding

Decreased plasma levels

Altered function

PATHOGENIC

Normal function

BENIGN

Figure 6 | Flow chart for the functional characterization of factor H (FH) variants. DAA, decay-accelerating activity; ELISA, enzyme-linked immunosorbent assay; FI, factor I.

Healthcare). Purity was confirmed by sodium dodecylsulfatepolyacrylamide gel electrophoresis–stained with Coomassie (Bio-Rad Laboratories, Hercules, CA) (Figure 1b and c). If no Y402H heterozygote carrier was available, the FH variant was purified together with FH wild-type protein. C3 was purified as previously described19 and cleaved to C3b by generating an AP C3 convertase.19 Factor B and factor I were isolated by immune-affinity chromatography. Concentration of purified proteins was assessed using absorbance at 280 nm, and molarities were calculated using the following extinction coefficients (mg1$ml 1 ): FH, 1.98; C3 and C3b, 0.98; factor B, 1.43; and FI, 1.53. FD was purchased from Calbiochem (San Diego, CA) and Millipore. Sheep red blood cell hemolytic assay The overall FH functionality was assessed using a modification of the FH-dependent SRBC hemolytic assay.20 The original assay was designed to test whether the serum from the aHUS patient lyses the sheep red cells. In our modified assay, we compared the amount of the FH variants and wild-type protein required to recover the regulatory capacity of a normal human serum that was depleted of 75% of the FH (NHSDFH). All experiments were done in triplicate. Blank was prepared by adding 20 mM ethylenediamine tetraacetic acid, and the maximum NHSDFH-induced lysis was established as 100% SRBC lysis. For positive and negative controls in our experiments, we used FH mutant protein with an altered C-terminal region and FH Kidney International (2018) 93, 470–481

polymorphic variants previously shown to not have functional impairment (Supplementary Figure S2). Decay-accelerating activity FH-dependent DAA was determined by surface plasmon resonance using a Biacore X100 (GE Healthcare). One thousand resonance units of C3b were immobilized on a CM5 chip, and AP C3 convertase was formed flowing 400 nM of factor B and 20 nM of FD in HEPES buffer with 5 mM MgCl2 and 0.005% Tween 20 for 150 seconds at a 30-ml/min flow rate. DAA of FH variants was determined flowing 70 nM FH diluted in HEPES buffer 0.005% Tween 20 for 300 seconds at a 10-ml/min flow rate. Wild-type FH (70 nM) was used as a reference sample. All assays were performed 3 times. Spontaneous decay of AP C3 convertase was determined by not injecting FH. FI cofactor activity The fluid-phase FI cofactor activity of FH was determined in a C3b proteolysis assay. Purified C3b, FH, and FI were diluted in HEPES buffer 0.02% Tween 20 at final concentrations of 170 nM, 25.8 nM, and 56.8 nM, respectively. Twenty microliters of mixtures were incubated in a 96-well plate at 37  C, and reactions were stopped at 2.5, 5, 7.5, and 12.5 minutes by adding 5 ml of 2% sodium dodecylsulfate, 62.5 mM of Tris, 10% glycerol, and 0.75% bromophenol blue. All assays were preformed twice. After centrifuging at 1800 rpm 479

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with SPSS software, version 21 (SPSS Inc., Chicago, IL). The Student t test for independent samples was used to compare the functional activities of the different FH variants with those of the FH wild type. Variation homogeneity between groups was confirmed by Levene’s test. P < 0.05 was considered statistically significant. DISCLOSURE

Conception, design, data collection, and analysis as well as the writing of this article were performed by investigators with no support from pharmaceutical companies. SRdeC has received honoraria from Alexion Pharmaceuticals for giving lectures and participating on advisory boards. None of these activities had any influence on the results or interpretations in this article. All the other authors declared no competing interests. The results presented in this paper have not been published previously in whole or part. Figure 7 | Model of the bent back structure of FH to illustrate the putative impact of the M832T gene variant. The overall structure of FH is based on known tilts and proposed side-by-side interactions as described.4,5 SCRs are represented by ovals, and the numbers of those relevant to the interaction with C3b and surface carbohydrates are indicated inside the ovals. The shaded circle indicates the interaction between SCR13 and SCR14 that is postulated here to be affected by the M823T gene variant. Carbohydrates binding to SCRs 20 and 7 are indicated as well as the thioester bond linking C3b to the surface (red dot). FH, factor H; SCRs, short consensus repeats.

for 5 min, samples were collected and analyzed in 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis. Gels were stained with Coomassie, and proteolysis of C3b was determined by measuring the cleavage of a0 -chain using a ChemiDoc Touch densitometer and the Image Lab software (Bio-Rad Laboratories). The C3b b-chain was used as an internal control to normalize the percentage of cleavage between samples. The percentage of cleavage was determined by the ratio a0 /b chains of C3b and setting as 0% the amount of a0 -chain at time 0. C-terminal C3b binding C3b binding by the FH C-terminal region was determined using a surface plasmon resonance strategy. Eight thousand resonance units of OX24, an mAb that binds to an epitope in the N-ter region of FH and blocks the interaction with C3b by this FH region, were immobilized on a CM5 chip. FH (10 nM) in HEPES buffer and 0.005% Tween 20, was flowed for 250 seconds and 500 seconds at 10 ml/min flow rate. Then C3b (200 nM) in the same buffer was flowed for 140 seconds at a 10-ml/min flow rate. All FH samples were assessed in triplicate. Differences in the capacity to bind C3b among the variants and the wild-type FH protein were determined by comparing the slopes of the linear representations that result from plotting the resonance units of captured FH versus the resonance units of C3b bound to the captured FH. In silico analysis of pathogenicity To functionally annotate the FH genetic variants detected in this study, we used ANNOVAR software,21 which provide a comprehensive bioinformatics analysis using different prediction algorithms. We considered likely pathogenic those variants in which a deleterious impact was anticipated by a majority of the prediction algorithms. Statistical analysis The normal range of FH plasma levels was determined using Reference Value Advisor c2.1.22 Statistical analyses were performed 480

ACKNOWLEDGMENTS

We are indebted to all patients and relatives participating in this study. SRdeC is supported by the Spanish Ministerio de Economía y Competitividad/FEDER (grant number SAF2015-66287-R), the Seventh Framework Programme European Union Project EURenOmics (grant number 305608), and the Autonomous Region of Madrid (grant number S2010/BMD-2316). SRdeC is member of the CIB intramural program Molecular Machines for Better Life (MACBET). This work was developed under the supervision of the Spanish Registry of the atypical Hemolytic Uremic Syndrome and C3 Glormerulopathy (aHUS/ C3G) registry. SUPPLEMENTARY MATERIAL Table S1. Allele frequencies of the CFH variants in disease and control populations. Table S2. Pathogenicity predictions. Figure S1. Plasma levels of the FH variants. (A) The allele-specific FH plasma levels for individuals who are heterozygotes for the Y402H polymorphism. The allele carrying the CFH variant is depicted in black. (B) The total FH levels for those CFH variants for which Y402H heterozygote carriers were not available. Normal range of expression for a single FH allele (50–127 mg/ml) and normal range for total FH levels (76–247 mg/ml) are indicated by a gray shadowed area. FH, factor H. Figure S2. Dynamic range of the sheep red blood cell hemolytic assay. The dynamic range of the SRBC assay was determined using factor H (FH) variants with known functional consequences. In agreement with early data, the 4 polymorphic variants 62V, 62I, 402Y, and 402H showed overlapping inhibition curves, with no significant differences between them in the amount of FH protein required to recover the complement regulatory capacity of the NHSDFH. Conversely, almost 8-fold more protein of a FH mutant lacking Cterminal functionality, FH-(1191L, 1197A), was required than normal FH to achieve 50% inhibition of the SRBC lysis. EC50 (50% effective concentration) value indicates the relative amount required to achieve the same protection as with wild-type FH. Supplementary material is linked to the online version of the paper at www.kidney-international.org. REFERENCES 1. Rodriguez de Cordoba S, Esparza-Gordillo J, Goicoechea de Jorge E, et al. The human complement factor H: functional roles, genetic variations and disease associations. Mol Immunol. 2004;41:355–367. 2. Sharma AK, Pangburn MK. Identification of three physically and functionally distinct binding sites for C3b in human complement factor H by deletion mutagenesis. Proc Natl Acad Sci U S A. 1996;93:10996–11001. 3. Meri S, Pangburn MK. Discrimination between activators and nonactivators of the alternative pathway of complement: regulation via Kidney International (2018) 93, 470–481

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