Deficiency of the Human Complement RegulatoryProtein Factor H Associated with Low Levels ofComponent C9

D. A. Falcao*, E. S. Reis*, D. Paixao-Cavalcante*, M. T. Amano*, M. I. M. V. Delcolli*, M. P. C.Florido*, J. A. T. Albuquerque*, D. Moraes-Vasconcelos�, A. J. Duarte�, A. S. Grumach� & L. Isaac*

Introduction

Factor H, first described as a b1-H-globulin [1], is a150-kDa glycoprotein and an important negative regula-tor of the complement alternative pathway. It is codedby a single gene (HF1) on human chromosome 1q32located within the regulators of complement activationgene cluster [2].

HF1 is closely linked to the FHR-1, FHR-2, FHR-3,FHR-4 and FHR-5 genes that code for five factor H-related (FHR) human plasma proteins [3]. The FHRproteins are composed of multiple repetitive domainstermed short consensus repeats (SCR) and are mainlysynthesized by the liver [4]. The HFI gene codes forfactor H and FHL-1. The latter is a 42-kDa proteinderived from alternative splicing of HF1. It is composedof the first seven N-terminal factor H SCR domains [5,6], is present in normal human plasma at a concentra-tion of 10–50 lg ⁄ ml and exhibits cofactor and decayaccelerating activities. Factor H is the best characterizedmember of this family and is composed of 20 SCR [7,8]. It is mostly secreted by the liver [9] and is presentin the serum of healthy adult individuals at a

concentration of 443 ± 106 lg ⁄ ml (normal range: 242–759 lg ⁄ ml [10]).

As a complement regulator, factor H competes withfactor B for C3b binding, preventing the formation ofthe alternative pathway C3-convertase. Factor H also pro-motes the dissociation of factor B or Bb from both C3-and C5-convertases of the alternative pathway [11–15].Moreover, factor H is the most important cofactor of theregulatory protein factor I in converting the C3b frag-ment into iC3b [16]. Factor H can also regulate the alter-native pathway activation on non-activating surfaces(such as self cells) by binding to surface sialic acid andglycosaminoglycans, such as heparin, heparin sulphateand dextran sulphate [17, 18]. It has been reported thatfactor H expression by some tumour cells [19] or factorH binding to some pathogenic microbes [20] can protectthose cells from complement activation. This protectionseems to be particularly relevant to host structures thatlack membrane-bound regulators, such as basement mem-branes in kidney glomeruli [21–23]. The role of the otherproteins of factor H family in controlling complementactivation is still under investigation; however, the highdegree of homology between these factor H-related

*Laboratorio de Complemento, Departamento

de Imunologia, Instituto de Ciencias

Biomedicas, Universidade de Sao Paulo, Brazil

and �Laboratorio de Investigacao Medica em

Dermatologia e Imunodeficiencias (LIM 56),

Faculdade de Medicina, Universidade de Sao

Paulo, Brazil

Received 11 January 2008; Accepted 11 June2008

Correspondence to: L. Isaac, Laboratorio de

Complemento, Departamento de Imunologia,

Instituto de Ciencias Biomedicas, Universidade

de Sao Paulo, Av. Professor Lineu Prestes

1730, CEP 05508-900, Sao Paulo, SP, Brazil.

E-mail: louisaac@icb.usp.br

Abstract

We identified a 4-year-old Brazilian boy from a family of Japanese descent andhistory of consanguinity, who suffered from severe recurrent pneumonia. Hecarries factor H (FH) deficiency associated with reduced levels of componentC9 and low serum levels of C3 and factor B. His mother also presented lowlevels of these proteins and factor I, while his father and sister had only lowerlevels of FH. Western blot assays confirmed the complete absence of FH andFHL-1 polypeptides in this patient. Sequencing of the proband’s FH cDNArevealed a homozygous G453A substitution, encoding an Arg127His change.His mother, father and sister are heterozygous for this substitution. Despitethe absence of FH in the plasma, this protein was detected in the patient’sfibroblasts, suggesting that Arg127 may be important for FH secretion. Lowconcentrations of C9 were detected in the proband serum but no mutations inthe patient’s C9 gene or promoter have been identified, suggesting that this isa consequence of uncontrolled complement activation and high C9consumption.

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proteins and factor H domains, SCR 6–10 and SCR 19–20, suggest they may present common functions [3, 4].

In agreement with its regulatory function, factor Habsence or dysfunction is associated with a higher suscep-tibility to diseases such as haemolytic uraemic syndrome(HUS) and membranoproliferative glomerulonephritis(MPGN) type II [23]. As the lack of factor H also corre-lates to low levels of C3, factor H deficiency can also beassociated to higher susceptibility to infection diseases[24]. Complete factor H deficiency is rare, as it has beendescribed in only 22 patients from 12 families, most ofwhich with a history of consanguinity (autosomal reces-sive inheritance pattern) [24].

C9 is the last component to be incorporated into themembrane attack complex, which is responsible for thelytic function of complement. C9 deficiency is relativelycommon in Japanese populations (1:1000) [25–27] butvery rare in Caucasians, with just few cases reported sofar [28, 29]. Although many Japanese C9-deficient indi-viduals are considered asymptomatic, there is evidencethat they carry a much higher risk for developing menin-gococcal meningitis than C9-sufficient individuals [30].

Here we report a case of factor H deficiency associatedwith reduced levels of C9 component. This is the firstfactor H deficiency case described in Brazil and, to ourknowledge, the first report where low levels of C9 havebeen correlated with a factor H deficiency. We investi-gated the molecular basis of the factor H and C9 defi-ciencies in this patient and screened for the presence andfunctionality of other complement elements in the pro-band’s serum.

Materials and methods

Proband. The proband (4 years old) is a male child froma family of Japanese descent and a history of consanguin-ity (his parents are cousins). At 2 years and 8 months, hedeveloped pneumonia with pleural effusion leading tohospitalization in the intensive care unit. Thoracic drain-age and intravenous antibiotics were introduced, improv-ing the respiratory insufficiency. The patient had hisimmunological profile evaluated presenting normal totalIgM, IgA and IgG subclasses levels (with the exceptionof IgG4 that was absent). Serum C3 was undetectableand he was referred to our outpatient group (Dr A. S.Grumach and Dr D. M. Vasconcelos). The patient pre-sented normal specific antibody production in response tovaccination against measles, rubella and decreasedresponses to non-capsulated Streptococcus pneumoniae andnormal lymphocyte proliferative response to mitogensand common antigens. Another lobar pneumonia wasdeveloped requiring hospitalization, and pneumococcalvaccination was repeated afterwards. The family hasanother child (6-year-old girl) who is healthy. Samples ofblood and skin biopsies were obtained with the parents

consent and this study was approved by our institutionalEthical Committee. The patient serum samples were col-lected when he was apparently healthy with no symptomsof infection.

Determination of haemolytic activity mediated by the classi-cal and alternative pathways. The haemolytic capacity ofthe proband and family members’ sera was determinedaccording to the modified agarose gel-diffusion protocol[31]. Serum samples (7.5 ll) were loaded into the geland incubated at 4 �C overnight, followed by incubationat 37 �C for 2 h to visualize the radial haemolysis aroundthe wells. The diameter of the lysis halo was determinedand expressed as a percentage of that observed using thepool of 47 normal human sera (NHS) using 100%, 75%,50% and 25% dilutions. The data were calculated usinglinear regression and accepted when R2 was ‡0.95.

Simple radial immunodiffusion assay. The levels of com-plement C3, C4, factor B, factor I and properdin in thepatient’s and his family sera were determined by simpleradial immunodiffusion assay [32].

Enzyme-linked immunosorbent assay. To measure factorH and C9 serum levels by enzyme-linked immunosor-bent assay (ELISA), 96-well plates were coated withrabbit anti-human factor H (3.4 lg ⁄ ml; kindly providedby Dr Sanchez-Corral-Hospital Universitario La Paz,Madrid, Spain) or goat anti-human C9 (1:1000 dilution;EMD Biosciences, Darmstadt, Germany) for 18 h at4 �C. Free sites were blocked with phosphate-bufferedsaline (PBS) containing 1% of BSA for 1 h at 37 �C.The wells were then loaded with different dilutions ofserum. Bound factor H or C9 were revealed using goatanti-human factor H (dilution 1:4000; EMD Bioscienc-es) or rabbit anti-C9 (dilution 1:4000; Serotec, Kidlington,UK) followed by alkaline phosphatase-conjugated anti-goat IgG or alkaline phosphatase-conjugated anti-rabbitIgG (dilution 1:5000; EMD Biosciences). Absorbancewas measured at 405 nm. Limit detection was0.24 ng ⁄ ml for factor H and 1.95 ng ⁄ ml for C9.

Western blot assay. Serum from the proband and hisfamily (diluted 1:75 in PBS) was separated by SDS-PAGE(10% acrylamide) and then electrotransferred to a nitrocel-lulose membrane. Free sites were blocked with TBSTbuffer [Tris–HCl (pH 8.0) 5 mM, NaCl 75 mM, Tween-200.028%] containing 1% of BSA overnight at 4 �C. Themembrane was then incubated with rabbit anti-humanfactor H (final dilution 1:5000) for 4 h at room tempera-ture. An additional blocking step was performed with 3%skimmed milk for another 18 h. Incubation with the alka-line phosphatase conjugated anti-rabbit IgG (1:10,000;EMD Biosciences) was then carried out for 1 h at roomtemperature. The membrane was then incubated with1 ml of nitroblue tetrazolium ⁄ 5-bromo-4-cloro-3-indolylphosphate (EMD Biosciences), and the reaction wasstopped with distilled water. Western blots to evaluate forfactor H protein family proteins were carried out using a

446 Factor H Deficiency and Reduced Levels of C9 D. A. Falcao et al...................................................................................................................................................................

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rabbit anti-human SCR 1–4 (kindly provided by Dr PeterZipfel, Department of Infection Biology, Leibniz Institutefor Natural Product Research and Infection Biology, Jena,Germany) as primary antibody using the same conditionsdescribed above.

The factor H secretion was studied using proband andnormal control fibroblasts (2 · 106 cells) in a 75-cm2 cellculture flask obtained after skin biopsies and cultured aspreviously described [33, 34]. The cells were stimulatedwith IFN-c (200 U ⁄ ml) for 6, 12, 24 and 48 h, and fac-tor H was subsequently evaluated in the supernatant andcell lysates from these cultures by Western blot assays.Samples were concentrated 40 times by lyophilization,protein concentration of each sample was determinedusing Bradford’s protein assay [35], and equivalentamounts of protein from the proband and normal controlsamples (supernatant: 2 ng ⁄ well and cell lysates:57 ng ⁄ well) were separated by SDS-PAGE and analysedby Western blot as described above. To evaluate serumC9, the primary and secondary antibodies used were goatanti-human C9 (1:5000) and alkaline phosphatase-conjugated anti-goat IgG (1:10,000) respectively (bothfrom EMD Biosciences).

Confocal analyses. These analyses were carried out asdescribed by Fishelson et al. [36]. Fibroblasts were stimu-lated with IFN-gamma (100 U ⁄ ml) for 24 h and thenfixed with 3.7% formaldehyde for 15 min. The cells weretreated with 0.2% Triton X-100 for 2 min and incu-bated with rabbit anti-human factor H or C9 polyclonalantibody (1:200) overnight at 4 �C. Cells were then incu-bated with anti-rabbit or anti-goat IgG conjugated withfluorescein isothiocyanate (Sigma, St Louis, MO, USA)for 1 h at room temperature, followed by the addition ofRNAse (10 mg ⁄ ml; Invitrogen, Carlsbad, CA, USA) andfurther incubation for 30 min. Cell nuclei were visualizedby incubation with propidium iodide and analysed usingan LSM 510-Zeiss confocal microscope (Carl Zeiss, Jena,Germany).

Total RNA extraction. Total RNA was extracted fromcultured fibroblasts using the Total RNA Isolation Sys-tem (Promega Co., Madison, WI, USA), following themanufacturer’s instructions. The total RNA concentrationwas determined by Gene Quant (GE Healthcare, Uppsala,Sweden).

cDNA synthesis and reverse transcription–polymerase chainreaction. Reverse transcription–polymerase chain reaction(RT–PCR) assays were carried out using the SuperScriptOne-Step RT–PCR System (Invitrogen), following manu-facturer’s instructions. First-strand synthesis was carriedout in a final volume of 50 ll containing 200 ng ofRNA, 10 mM dNTP, 5 ll of 10· buffer, 20 pmol ofeach primer (Table 1) and 5 U of RT ⁄ Taq mix incubatedfor 30 min at 50 �C. DNA was denatured at 94 �C for2 min followed by 40 cycles carried out as follows:1 min at 94 �C, 1 min at 55 �C and 1 min at 72 �C. A

final amplification step was carried out at 72 �C for7 min. The products were analysed by gel electrophoresisand staining with ethidium bromide.

PCR of genomic DNA. DNAzol (500 ll; Invitrogen)was added to approximately 2 · 106 fibroblasts. Ethanol100% (300 ll) was added and the solution was centri-fuged at 10,000· g for 10 min. The pellet was washedwith 1 ml of 95% ethanol and resuspended in 200 ll of10 mM Tris–HCl (pH 7.5), 0.1 mM EDTA.

Polymerase chain reactions were carried out as 50 llreactions containing 100–300 ng of genomic DNA, in20 mM Tris–HCl (pH 8.4), 50 mM KCl, 5 mM MgCl2,10 mM dNTP, 50 ng of each primer and 5 U of DNApolymerase. PCR reactions were performed for 40 cyclesas follows: 1 min at 95 �C, 1 min at 50–62 �C and2 min at 72 �C. A final extension step for 7 min at72 �C was included. PCR was used to amplify factor Hexon 4 (Table 1) and all C9 gene exons (Table 2).

Sequencing. Amplified genomic DNA and cDNAproducts were sequenced using the ABI PRISM BigDyeTerminator Cycle Sequencing Ready Reaction (AppliedBiosystems, Foster City, CA, USA). The nucleotidesequences were compared with the human factor HcDNA sequence (accession number NMY00716) andhuman C9 gene (acession number NM001737.2) accord-ing to Altschul et al. [37].

Results

Functional evaluation and levels of complement proteins

The proband’s serum presented undetectable haemolyticactivity mediated by classical or alternative pathways(Table 3). These observations are consistent with ourinability to detect C3 in the his serum (Table 3). Factor

Table 1 Sequence of oligonucleotides used for factor H RT–PCR and

PCR.

Exons-specific

numbering

positions Sequence 5¢–3¢ Sense

RT–PCR

1 ATT TCT TGG AAG AGG AGA AC F

753 ATA AGG AGA ATG AAC GAT TTC F

1483 TTT TAA GGC ATA TGT ATA CGT R

1560 AAG ATG GAT GGT CAG CTC AAC F

1662 TCA TTC AGC TTA AAC CAT GTG R

1969 ACC TCC TGA ACT CCT CAA TG F

2698 CTG AGG TGG TTG TGA ACA TG R

3167 TGC ATT AAT AGC AGA TGG AC F

3186 GTC CAT CTG CTA TTA ATG CA R

3919 CCA CCG GTC TCA GCT TAT AA R

PCR

FH GEN F CAG TCC ATG CAC CAA GAA GGA F

FH GEN R CAG GCT GCA TTC GTT TTT GGC R

F, forward; R, reverse.

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I and properdin were present in the normal range. How-ever, we also detected reduced levels of factors B and H(Table 3). These data are compatible with a secondary C3

deficiency caused by the impairment in the regulation ofthe alternative pathway.

We also evaluated haemolytic activity and comple-ment levels in other members of the family. We observednormal levels of complement-dependent haemolytic activ-ity in the father’s serum and lower factor H levels(190.9 lg ⁄ ml) than observed in healthy adults(443 ± 106 lg ⁄ ml; normal range: 242–759 lg ⁄ ml [10])(Table 3). In the mother’s serum, we also found approxi-mately 50% (140.5 lg ⁄ ml) of normal factor H concen-trations and low levels of C3, factors I and B. Thereduced levels of alternative pathway proteins andreduced C3 are probably responsible for the impairedactivity of alternative pathway found in her serum(Table 3). The patient’s sister’s serum also presentedfactor H levels corresponding to approximately 50%(143.6 lg ⁄ ml) of normal levels and complement-dependent haemolytic activity was in the normal range(Table 3). These data indicate a factor H deficiency inthis family. The observations that the parents presentedfactor H at around 50% of normal levels suggest an auto-somal recessive pattern of inheritance.

We also observed that serum C4 levels were higher inall the family members except for the mother, who dis-played normal C4 levels (Table 3). An evaluation of theterminal pathway components (C5–9) in this familyrevealed that the proband’s serum had significantlyreduced levels of C9. The proband’s serum presented only17.5% (5.6 lg ⁄ ml) of normal C9 levels established inour laboratory for 30 Brazilian healthy adults donors(28.5 ± 6.5 lg ⁄ ml), while his parents and sister pre-sented normal C9 levels (Table 3).

Table 3 Complement mediated haemolytic activity and protein levels in patients’ and family members.

Patient Father Mother Sister Normal range

CPH (%) 0 127.59 77.25 119.67 56–192

APH (%) 0 117.5 49.21 82.2 71–171

C3 (lg ⁄ ml) << 963.5 382.6 1271.1 3–4 years old: 905 (range: 610–1410) [49]

5–6 years old: 1075 (range: 770–1390) [49]

Adults: 800–1900 [50]

C4 (lg ⁄ ml) 467.7 807.3 514.4 615.5 3–4 years old: 164 (range: 95–302) [49]

5–6 years old: 177 (range: 101–309) [49]

Adults: 450–600 [50]

Factor H (lg ⁄ ml)* 16.8 190.9 140.5 143.6 1–5 years old: 606 (range: 233–1319) [10]

6–12 years old: 684 (range: 225–1636) [10]

Adults: 443 (range: 242–759) [10]

Factor I (lg ⁄ ml) 51.6 52.2 23.6 ND 1–5 years old: 58 (range: 30–104) [10]

6–12 years old: 57 (range: 34–91) [10]

Adults: 64 (range: 39–100) [10]

Factor B (lg ⁄ ml) << 105.9 62.4 ND 3–4 years old: 34 (range: 20–59) [49]

5–6 years old: 40 (range: 25–60) [49]

Adults: 74–286 [51]

Properdin (lg ⁄ ml) 12 ND ND ND 1–5 years old: 23 (range: 13–38) [10]

C9 (lg ⁄ ml)* 5.6 38.5 33.5 30 2–18 years old: 59 [52]

Adults: 22–35

<<, below detection limits; ND, not determined.

*Determination by ELISA. All other protein concentrations were determined by immunodiffusion.

Table 2 Sequence of oligonucleotides used for C9 PCR assays.

Exons-specific

numbering

positions Sequence 5¢–3¢ Sense

1F ATT CTC CTT TGG TTG GAC TTC F

1R CAT TGT CAT GTA CTT TGC TGC R

2F GAC TTC TTG GAA ACA GAT ATA C F

2R GGC TCC CTG CCT CAT AAT CAT R

3F AAC CAT TGA CTG ATT GCA GGG F

3R TTC ATT CAG GAA GGC ACA CAG R

4F GAT ACC TCA CCT CCA GGG TTA F

4R CAC CTA TGT CCC TCG CAC AAA R

5F GTT GTG GTA TTT CCA CTT CTG F

5R TCC AAA CTA CAT CGC CTC TTC R

6F TTG CTC TTA ACT CCT TTG TTC F

6R TGG GTA GTT TGG AAC CTT TC R

7F GGT ATC TCA TGG TCA CTG CTA F

7R GCA GGT TAC AGG GCT TTG TAT R

8F CAG GAC AGA CAT GAT GGC ACA F

8R CAT TGA CAT CTA CCC TCA GGC R

9F CTT TTG ATA ACT GGC TTC TC F

9R AAT GAC ACA GTC TTC TGT TTG R

10F GTA TTC TAC AAG TTC TCT CAG F

10R AAT TAG ATA ACC CCA AAG TGC R

11F GGA CCC AAA GTA AAA TCC TAT TG F

11R CTG AAT GAA TGT ATG CAC ACC R

P1F GTT GTG ATG TCC ACA TTC TCT F

P1R GAC ATG CTG CTC TTG CTG GGT R

P2F CTT GTT GCC CAG GCT GGA GTT F

P2R ATG GTG GTG GGT GCC TGT AAT R

F, forward; R, reverse; P, promoter.

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Evaluation of serum factor H family proteins

We analysed the profile of factor H family proteins pres-ent in the sera of the proband and his family members byWestern blot (Fig. 1). The 150 kDa band correspondingto full-length factor H was present in his sister’s and par-ents’ sera but was completely absent in the serum of theproband, confirming his complete deficiency. We observedtwo bands of approximately 37 and 42 kDa in the pro-band’s serum, which correspond to the sizes of FHR-1a(37 kDa) and FHL-1 (42 kDa) and ⁄ or FHR-1b (43 kDa)respectively. A non-specific 116-kDa band was detectedin the parents’ and sister’s sera using rabbit anti-humanfactor H. To confirm these observations, a second set ofWestern blot analyses was carried out in which weemployed an antibody that reacts with SCR 1–4 from fac-tor H and FHL-1 but does not react with FHR-1b(Fig. 2). The father’s, mother’s, sister’s and NHS samplespresented the corresponding 150 and 42 kDa bands and,in accordance with previous results, no factor H wasobserved in the proband’s serum (Fig. 2). Furthermore,the FHL-1 42-kDa band was also completely absent inthe proband’s serum, indicating the absence of both factorH and FHL-1 in the serum from this patient. These anti-bodies also detect unidentified 65 and 86 kDa proteins inall the sera evaluated (Fig. 2). As FHR-5 is a 65 kDa andFHR-4A an 86 kDa protein, this rabbit anti-human fac-tor H antibody may also be reacting with these proteinsbelonging to the factor H family.

Evaluation of serum C9

Because of the proband’s Japanese background and ourability to detect only low amounts of C9 by ELISA, wedecided to evaluate the C9 protein profile in all familymembers’ sera by Western blot. Fig. 3 shows that theproband’s serum presented a very faint 70-kDa band

corresponding to full-length C9 when compared to thatobserved for his parents and sister.

RT–PCR of factor H cDNA

Using total RNA from proband’s family and normal con-trol fibroblasts, we amplified several regions that span thefull length of the factor H cDNA. No differences in sizeand intensity were found after comparison of the amplifiedproducts from the proband and control samples (data notshown). Sequencing of these fragments revealed a homozy-gous single nucleotide substitution (CG453T fi CA453T)in the patients’ cDNA generating an Arg127His modifica-tion in SCR-2, which is present in both factor H and FHL-1 proteins (Fig. 4A). The mother (Fig. 4B), the father(Fig. 4C) and the sister (data not shown) are heterozygousfor this mutation. The presence of this mutation wasconfirmed in the genomic DNA from the patient and hisparents.

We also detected two other mutations in the patient’scDNA: a silent CAA2089 fi CAG2089 mutation in theGlu672 codon in SCR-11 and a GAG2881 fi GAT2881

substitution, generating a Glu936Asp mutation in SCR-15. Both mutations have been described as polymor-phisms [38, 39] and apparently do not, on their own,lead to deleterious consequences in protein function.

Sister Mother Father Patient Normal

150 kDa

116 kDa

42 kDa

37 kDa

Figure 1 Western blot analysis of factor H and proteins from factor H

family present in the proband, family members’ and control sera. All

samples were used under reduced conditions. Similar results were

obtained in three independent experiments.

SisterMotherFatherPatientNormal

FH

FHL-1

Proteins Proteins

150 kDa

~86 kDa

~65 kDa

42 kDa

Figure 2 Western blot analysis of factor H and FHL-1 proteins in the

proband, family members’ and control sera after SDS-PAGE under

reduced conditions. The rabbit polyclonal anti-human factor H recog-

nizes SCR 1–4. Proteins refer to purified factor H. Similar results were

obtained in three independent experiments.

Sister Mother FatherFather (20) Patient Normal

~70 kDa

Figure 3 Western blotting analysis of C9 protein profile present in the

proband, family members’ and control sera after SDS-PAGE under

reduced conditions. All sera samples were used at 1:10 dilution, with

exception of father (20) – diluted at 1:20. Similar results were obtained

in three independent experiments.

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PCR of the C9 gene

Using proband’s and controls’ genomic DNA, we ampli-fied the 11 exons of the C9 gene including intron ⁄ exonboundaries. No size differences between the proband’sand control fragments were detected (data not shown).The proband’s fragments were sequenced and the onlydifferences observed were three previously described poly-morphisms [40]: (1) a G fi A substitution 10 bp fromthe end of intron 1; (2) a CC17G fi CT17G substitu-tion in exon 1, generating an Arg5Trp mutation; and (3)a silent AAC1975 fi AAT1975 substitution in exon 11.As no other mutations that could be implicated in theproband’s C9 deficiency were detected, we decided tosequence the promoter region of the C9 gene. Sequencingof a 1000-bp stretch of the promoter region failed toreveal any alterations.

Confocal analysis of factor H and C9 production by proband

fibroblasts

The only novel change in the proband’s DNA thatcould possibly explain the factor H deficiency was the

homozygous single nucleotide substitution(CG453T fi CA453T) leading to the Arg127His modifi-cation. One hypothesis is that this mutation may affectfactor H and FHL-1 stability and ⁄ or secretion. To testthis hypothesis, we used confocal microscopy to evaluatethe production of factor H epitopes in the cytoplasm ofthe proband’s fibroblasts. Fig. 5 shows that a moreintense signal corresponding to factor H epitopes wasdetected in the proband’s cells than in control fibroblasts.This result indicates that proband’s cell are capable ofproducing factor H epitopes that are not secreted butinstead accumulate in intracellular compartments. Thesame analysis was carried out on fibroblasts of othermembers of the proband’s family and all cells showed anintensity signal similar to that of the normal control(data not shown).

We then used confocal microscopy to evaluate thepresence of C9 epitopes within the fibroblasts. Eventhough the proband’s fibroblasts showed a less intensesignal for C9 epitopes when compared to control’s cells(Fig. 6), this qualitative method does not on its ownallow us to conclude that the patient’s cells are producingsignificantly fewer C9 molecules.

Figure 4 Sequencing results from the pro-

band and parents’ factor H cDNA. Chroma-

tographs from proband (A), his mother (B)

and father (C) showing the CG453T fiCA453T substitution, which results in a

R127H residue change.

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Factor H secretion profile by proband’s fibroblasts

We cultured proband’s and control’s fibroblasts and eval-uated the presence of factor H in the cell lysates and su-pernatants following 6, 12, 24 and 48 h of stimulationwith IFN-c (200 U ⁄ ml). The 150-kDa band (factor H)was present in proband fibroblast supernatants (secreted)at very low levels when compared to control’s supernatant(Fig. 7). However, the cell lysates (intracellular) alreadyexhibited the factor H protein after 6 h of culturingstimulus and this accumulation increased after 12, 24and 48 h of IFN-c stimulation. Factor H was practicallyabsent in the normal cell lysates, although it was possibleto visualize factor H at all the times in the supernatants(Fig. 7). When we investigated the production of factorH by the mother’s fibroblasts. we observed that her cellswere capable of secreting this regulatory protein,although at levels lower than that observed for the nor-mal control (data not shown).

Discussion

Genetic deficiencies of complement proteins, includingeffectors and regulators, have been already described,most of them related to high susceptibility to infectionsor autoimmune disorders such as systemic lupus

erythematosus and glomerulonephritis [24, 41]. Here wedescribe the first case of factor H deficiency in a Brazilianpatient. Interestingly, this factor H deficiency was associ-ated with a partial deficiency in complement C9. To ourknowledge, this combination of complement deficiencieshas not been described in the literature.

Complete factor H deficiency is a rare phenomenon(only 22 cases from 12 different families) [24, 41–43]

and restricted data are available regarding geneticmutations leading to factor H deficiency. Ault et al.[41] evaluated a factor H-deficient patient and identi-fied mutations in the codons for two cysteine residues:one in SCR-9 and the other in SCR-16. Both muta-tions, T1679C (Cys518Arg) and G2949A (Cys941Tyr),modify conserved Cys residues that form disulfidebonds responsible for the maintenance of the SCRdomain tertiary structure. In a subsequent study, thesame group showed that the C518R and C941Y mutantswere retained in the endoplasmic reticulum in trans-fected cells. This suggests that the factor H deficiencyin this patient was in fact due to impaired factor Hsecretion [44].

In another study, three Italian factor H-deficientpatients were shown to be homozygous for a nonsensemutation, G638T, generating a Glu171Stop. As Glu171 islocated within SCR-3 of factor H and FHL-1, this

A

C D

B

Negative control FH

Figure 5 Confocal analysis of factor H epi-

topes present in proband and normal control

fibroblasts. Normal control (A,B) and pro-

band (C,D) were visualized with propidium

iodide (nuclei, in red), rabbit anti-human fac-

tor H antibody (primary antibody) and goat

anti-rabbit IgG antibody (B,D) (in green) or

with propidium iodide and anti-rabbit IgG

only (A,C).

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mutation resulted in the complete absence of both pro-teins in the patients’ plasma [45].

Patients with factor H deficiency have clinical aspectssimilar to a secondary and acquired C3 deficiency inwhich C3 levels are reduced to varying degrees, fromundetectable to approximately 50% of normal [24]. Thesepatients may also present factor B, C5 and properdinbelow normal levels, which reflects uncontrolled alterna-tive pathway activation. As a result, they present highersusceptibility to infections by Streptococcus pneumoniaeand Neisseria meningitidis or immune complex deposition-

mediated diseases such as systemic lupus erythematosusand MPGN. Patients with partial factor H deficiencyhave been associated with a higher risk of developingHUS [24].

Due to our inability to detect factor H (150 kDa) andFHL-1 (42 kDa) in the proband’s serum by Westernblot, we hypothesized that the responsible mutationwould be located within the first seven SCR of those pro-teins. This prediction was confirmed when we sequencedthe proband’s factor H cDNA and detected a singlenucleotide substitution, G453A within exon 4 that gen-erated a novel Arg127His substitution within SCR-2 offactor H and FHL-1. Dragon-Durey et al. [43] also foundan alteration in the same residue (G453T, generating anArg127Leu) in two HUS patients (brothers) with <1% ofthe normal factor H concentration. They attributed thefactor H deficiency to this mutation [43], which was notdetected in the factor H genes of 200 normal subjects,indicating that it is unlikely to be a polymorphism (DrM. Dragon-Durey, pers. comm.). The mutated residueArg127 is highly conserved and is in close vicinity to oneof the highly conserved Cys residues important for themaintenance of SCR-2 tertiary structure (Table 4). So,this mutation may alter factor H and FHL-1 tertiarystructure or stability and consequently its secretion asdemonstrated in Fig. 7. This factor H deficiency was

Stimulationtime (h)

Normal

Lysate

6 12 24 48 6 12 24 48

150 kDa

150 kDa

Supernatant

Patient

Figure 7 Secretion of factor H by fibroblasts. Cells from the patient

and the normal control were treated with IFN-c for 6, 12, 24 and 48 h.

Cell lysates and supernatants were harvested, concentrated and equal

amounts of total protein were loaded in SDS gel before electrophoresis.

Similar results were obtained in three independent experiments.

A B

C D

Negative control C9

Figure 6 Confocal analysis of C9 present in

proband and normal control fibroblasts. Nor-

mal control (A,B) and proband (C,D) were

visualized with propidium iodide (nuclei, in

red), goat anti-human C9 antibody (primary

antibody) and rabbit anti-goat IgG antibody

(B,D) (in green) or with propidium iodide

and anti-goat IgG only (A,C).

452 Factor H Deficiency and Reduced Levels of C9 D. A. Falcao et al...................................................................................................................................................................

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Journal compilation � 2008 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 68, 445–455

inherited by an autosomal recessive pattern as the parentspresented approximately 50% of normal factor H serumlevels. This correlates with the observation that both par-ents are heterozygous for the Arg127His substitution.Future studies should address the role of Arg127 in thesecretion of factor H.

We also observed low levels of C9 in this patient.Even though the proband’s family is of Japanese descent,he did not carry the C343T substitution that is com-monly found in C9-deficient Japanese individuals.Horiuchi et al. [25] analysed the molecular basis of 10unrelated Japanese C9-deficient patients and showed thatall had a C343T substitution that created a prematurestop codon at position 95 (Arg95) and one had an addi-tional amino acid substitution at 507 position (C507Y).The authors suggested that because of the high preva-lence of the homozygous Arg95Stop mutation among Jap-anese subjects (1:1000), it was probably present in thefounding individuals of this population, as has been sug-gested for the common C2 deficiency in the Europeanpopulation. Orren et al. [46] also reported a mutationresponsible for the replacement of a cysteine by a glycineat position 98 in the C9 protein from an Irish family.This alteration leads to severely reduced serum C9 (0.2%of normal concentration).

The above-mentioned mutations were not present inthe patient’s C9 gene nor were any alterations identifiedin the promoter region. Therefore, one possible explana-tion for the reduced C9 levels found in the patient’sserum could be excessive activation of the alternativepathway as a consequence of factor H deficiency. Onemay consider that the low levels of C9 in the patient’sserum could be attributed to his early age (4 years old)as it is well known that C9 serum levels are very low at

birth [47]. However, this does not appear to explain thepresent case as the proband’s 6-year-old sister presentedC9 levels comparable to that observed in normal adults.Therefore, we believe that the low levels of C9 in theproband’s serum could be related to greater consumptionof complement proteins, and is not due to the inherentlylow C9 levels found in children. This hypothesis is inagreement with observations by Levy et al. [48] whodetected decreased levels of terminal pathway components(C5, C6, C7, C8 and C9) in two Algerian brothers pre-senting factor H deficiency.

Acknowledgment

We thank Dr Noac Chuffi Barros and Dalton Bertolinifor the clinical follow-up and Chuck S. Farah for review-ing the manuscript. This work was supported byFundacao de Amparo a Pesquisa do Estado de Sao Pauloand Conselho Nacional de Pesquisa e DesenvolvimentoTecnologico, Brazil.

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