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Clinical Immunology (2009) 131, 456–462

Properdin deficiency associated with recurrent otitismedia and pneumonia, and identification of malecarrier with Klinefelter syndromeLone Schejbel a ,⁎, Vibeke Rosenfeldt b, Hanne Marquart a,Niels Henrik Valerius b, Peter Garred a

a Department of Clinical Immunology, Copenhagen University Hospital, Rigshospitalet, Denmarkb Department of Pediatrics, Copenhagen University Hospital, Hvidovre, Denmark

Received 12 November 2008; accepted with revision 14 February 2009Available online 27 March 2009

⁎ Corresponding author. Department7631, Rigshospitalet, Blegdamsvej 9, 235398766.

E-mail address: l.schejbel@gmail.c

1521-6616/$ – see front matter © 200doi:10.1016/j.clim.2009.02.008

KEYWORDSProperdin;PFC;Deficiency;Alternative complementpathway;Meningococcal disease;Immunodeficiency;Otitis media;Pneumonia;Klinefelter syndrome

Abstract Properdin is an initiator and stabilizer of the alternative complement activationpathway (AP). Deficiency of properdin is a rare X-linked condition characterized by increasedsusceptibility to infection with Neisseria meningitidis associated with a high mortality rate. Wereport properdin deficiency in a large Pakistani family. The index cases were found by screeningfor immunodeficiency due to a history of recurrent infections. This revealed absent AP activity,but normal classical and lectin pathway activity. Sequencing of the properdin gene (PFC)revealed a novel frameshift mutation. When all available relatives (n=24) were screened for themutation, four affected males, four female carriers and a male heterozygous carrier wereidentified. He was subsequently diagnosed with Klinefelter syndrome. A questionnaire revealed astriking association between properdin deficiency and recurrent otitis media (P=0.0012), as wellas recurrent pneumonia (P=0.0017).This study is the first to show a significant association between properdin deficiency and

recurrent infections.© 2009 Elsevier Inc. All rights reserved.

of Clinical Immunology Sect100 Copenhagen O. Fax: +45

om (L. Schejbel).

9 Elsevier Inc. All rights reser

Introduction

Properdin is a basic glycoprotein, synthesized by peripheralblood T-cells, monocytes and neutrophils [1–3], and normallypresent in serum at concentrations between 11–37 mg/l [4].

Patients with properdin deficiency have a 250-fold higherrisk of meningococcal disease than the general population

ved

[5]. The mean age at onset of symptoms is 12–14 yearsand the mortality rate from meningococcal disease is high(33–50%), but recurrent disease is uncommon in contrastto patients with late complement component deficiencies[5–7]. Within a family, properdin deficient relatives have 18%risk of meningococcal disease, and the susceptibility todisease may be promoted by other factors in the immunesystem such as MBL-deficiency or lack of the IgG2 allotypemarker G2m(n) [4,5,8].

Properdin is essential for efficient activation of comple-ment via the alternative activation pathway (AP): In the

.

457Properdin deficiency is associated with recurrent otitis media and pneumonia

standard version of AP activation C3 is spontaneously cleavedto C3a and C3b. C3a functions as a chemo attractant whileunspecific binding of C3b to foreign surfaces facilitates thebinding of factor B. Cleavage of factor B and release of Bagenerates the AP C3 convertase complex C3bBb, whichaccentuates C3 cleavage and activation of the downstreamcomplement cascade. Binding of properdin to C3bBb isessential for efficient AP activation; it stabilizes theotherwise labile complex, extending the half-life up to10-fold [9]. Moreover, recent studies have suggested a moreactive role of properdin as an initiator of the AP pathwayand are central for the conceptual understanding ofproperdin deficiency in relation to disease [10–12]: proper-din binds to bacterial surfaces – probably as an innatepattern recognition molecule – and promotes C3b-deposi-tion and formation of the C3bBbP-complex. Indeed, bacter-ial surfaces that bind properdin activate AP rapidly whilebacterial surfaces that do not bind properdin activate APslowly [12]. The surface of Neisseria strains containslipooligosaccharide (LOS). C3b only binds to the surface ofNeisseria gonorrhoeae if it is pre-treated with properdin[12], and it has been shown that LOS-induced systemiccomplement activation does not occur in serum fromproperdin deficient mice, suggesting that complementactivation through the alternative pathway is essential forcomplement activation in response to Neisseria [11].

The first description of properdin deficiency was inpatients from Sweden in 1982 [13]. Since then, genetic ana-lyses have shown a great heterogeneity among 24 familieswith identification of 16 different mutations in the Properdingene (PFC) [14,15]. The inheritance of properdin deficiencyis X-linked recessive: PFC is located on Xp11.3–Xp23 andconsists of 10 exons covering approximately 6 kb [16–18].The monomeric properdin molecule consists of six repetitiveType-I repeat sequence (TSR)-like domains surrounded by theN- and C-terminal regions [19]. The monomers oligomerize inhead to tail orientation into di- tri- and tetrameric moleculeswhich are present in serum in an approximate ratio of 1:2:1[20,21]. Properdin deficiency has been divided into threecategories; tpI with no (b0.01 mg/l) properdin in serum, tpIIwith b10% properdin in serum, and tpIII with normal concen-tration but dysfunctional properdin in serum. TpI properdindeficiency is caused by nonsense or missense mutations thatmay interfere with oligomerization of properdin in di- tri-and tetramers. mRNA transcription is normal in cells of tpIdeficient patients but no protein is detected probably due torapid intracellular degradation [22]. All nonsense mutationsdescribed so far result in truncated proteins that lack TSR6which has been shown to be necessary for oligomerization[23]. In tpII deficiency, properdin is synthesized and secretedfrom the cells, but oligomerize in an abnormal pattern with adominance of dimers. The reduced properdin concentrationin tpII deficiency is probably due to rapid extracellulardegeneration of abnormal properdin molecules [22]. Onlyone case of properdin deficiency tpIII has been described asyet. This was caused by a missense mutation, which affectedthe binding of properdin to C3b [24].

Most of the properdin deficient patients described so farhave been Caucasian of origin, either due to an over-representation of properdin deficiency among Caucasians ordue to insufficient research of properdin deficiencies inother populations [14].

Here we describe the genetic background of properdindeficiency in a family of Pakistani origin. The geneticanalyses identified four properdin deficient patients includ-ing a son of an unexpected female carrier, and surprisingly amale carrier with Klinefelter syndrome was found. Prior tothe genetic analysis a structured interview regardingprevious infections was performed. This revealed an associa-tion between properdin deficiency and recurrent otitismedia, as well as pneumonia.

Materials and methods

Patients

Twenty-four members from three generations of a Pakistanifamily were included in the study after informed consent.The grandparents in the family (I:1,1:2) are ancestors of anIndian family, who fled to Pakistan during the conflictbetween Kashmir and Punjab. In 1973 they (I:1,1:2)immigrated to Denmark with their oldest children. Theiryoungest daughter (II:2) and their grandchildren were born inDenmark.

Analysis of the complement system

Screening of the classical, alternative and lectin comple-ment pathways was performed using the ELISA system,“Wielisa”, as recommended by the manufacturer (Wieslab,Sweden). In short, the wells of microtiter strips for classicalpathway evaluation were pre-coated with IgM, strips foralternative pathway determination were coated with LPS,and MBL pathway strips were coated with mannan. To ensurethat only the intended pathway was activated at eachmicrotiter strip, sera were diluted in buffers containingspecific blockers of the other complement-activating path-ways. Complement activation was detected by measuringthe amount of C5b–9 terminal complex (TCC), with anantibody against a neoepitope expressed during TCC forma-tion [25].

Detection of properdin was performed using immunoelec-trophoresis and ELISA as described previously [26].

Questionnaire

All family members (or their parents) were questioned in astructured interview regarding the occurrence and fre-quency of previous infections including otitis media, pneu-monia, meningitis, septicemia or any other severeinfections. Hospital records were reviewed for all cases ofprevious hospitalizations for infectious disease.

Sequencing of the PFC gene

DNA was extracted with the Promega Maxwell system asinstructed by the manufacturer. All exons and the splicesignals of the properdin gene were sequenced in patient III:1.In the rest of the family exon 5 was sequenced. Primers forPCR-amplification and sequencing are listed in Supplemen-tary Table 1. Sequencing was done with BigDye Terminatorsequencing kit 3.1 (PE Applied Biosystems, CA, USA).

458 L. Schejbel et al.

Sequence alignment and comparison to GenBank sequenceNM002621 was done with BioEdit Sequence Alignment Editorversion 7.0.1 (freeware: http://www.mbio.ncsu.edu/BioE-dit) [27].

Results

Case studies

Case 1+2:. The index patients (III:1+2) were two identicaltwins with consanguineous parents of Pakistani origin. Theywere born at the gestational age of 33 weeks by electivecaesarean section due to intrauterine growth retardation intwin A.

Patient III:1. Twin A, birth weight 1600 g, was treatedwithin the first week for suspected septicemia. Bloodcultures for bacterial pathogens were negative, and therecovery was uneventful. From the age of 15–16 months, hehad been ill every other month with recurrent otitis media,upper and lower respiratory tract infections, diarrhea andpoor weight gain. In his second year he was admitted twicewith high fever and a clinical and laboratory suspicion ofsepticemia. On both occasions the CRP was above 150 mg/Land neutrophil count was significantly elevated. Heresponded rapidly when treated with cefuroxime andgentamycin. Blood cultures were negative.

Patient III:2. Twin B, birth weight 1852 g, had anuncomplicated neonatal period. Like his twin brother, hehad recurrent respiratory infections, otitis media anddiarrhea, which started in his first year. When 2 years old,he was admitted after a prolonged fever of 2 weeks andlaboratory evidence of a bacterial infection with CRP 190 mg/L and total white blood count 32.7×109/L, neutrophils28.0×109/L. Chest X ray showed a right upper lobe infiltratewith atelectasis. Blood cultures were negative, and cultureof sputum exhibited Haemophilus influenzae and Moraxellacatarrhalis. He responded well to cefuroxime.

The increased infection rate, as well as the fact that theparents were cousins raised the suspicion of a geneticimmunodeficiency, and the patients were referred toimmunological investigation. Analysis of complement acti-vation with Wielisa showed a decreased activation ofcomplement through the alternative activation pathway tob1% in both patients (normal range 30–113%). The activitiesof the lectin and classical pathways were normal. Furtherevaluation of the complement system in patient III:1 showeda decreased level of properdin to 10% and 16% of normalvalues in two independent samples analyzed by electro-immunoassay, leading to the diagnosis of properdin defi-ciency. All of the other evaluated immunological parameterswere normal including: concentration of C3, C4 and C1q;Immunoglobulin concentrations (including IgG subclasses);fraction and concentration of T-, B-, NK cells (andsubpopulations); expression of adhesion molecules; prolif-erative response of lymphocytes to stimulation with PHA,ConA and PWM; MBL-genotype (XA/YA), and MBL-concentration.

Case 3. Simultaneouslywith the immunological evaluation ofpatients III:1+2, their 16-year old cousin (III:6) was admittedwith high fever for 2 days, severe headache, confusion andpetecchiae. Lumbar puncture showed leucocytes 1236×106/L.

Activated partial thromboplastin time, prothrombin time andD-dimer were elevated. He was immediately treated with i.v.penicillin and corticosteroids. He was also intubated and kepton assisted ventilation for 2 days. Culture of cerebrospinalfluid was positive for Neisseria meningitidis, serotype Y. Herespondedwell to treatment and recovered without sequelae.

During the diagnostic work-up his past medical history wasfurther explored. This revealed no previous hospitalizations,but the patient reported recurrent stomatitis every thirdmonth, recurrent otitis media, and a previous episode of asubcutaneous abscess.

Evaluation of the complement system showed b2%activation via the alternative pathway.

Case 4. A two-year old boy (III:13), identified as properdindeficient during the investigation of the family. Evaluation ofthe complement system showed b2% activation via thealternative pathway. His medical history revealed 2 episodesof otitis media, one lower respiratory infection, and oneepisode of prolonged gastroenteritis for which he washospitalized. He had experienced no serious invasiveinfections.

All family members with properdin deficiency havereceived quadrivalent polysaccharide meningococcal vac-cine and polyvalent pneumococcal vaccine. They have beenprovided oral penicillin and have been instructed to take thisin case of fever above 38.5 °C, and to seek immediatemedical attention.

Retrospective evaluation of infections in all familymembers

All family members underwent a structured interviewregarding previous infections. This interview was per-formed before the results of genetic evaluation wereavailable. Specifically, each family member was asked ifhe or she ever, or, – if confirmed – at which age and howoften, had experienced otitis media, pneumonia, meningi-tis, septicemia or any other severe infections. Results areshown in Table 1. No difference in the infection tendencybetween non-carriers and heterozygous carriers was found.All four properdin deficient patients reported severalepisodes of otitis media while only two of the 21 family-members had experienced (recurrent) otitis media(P=0.0012, Fishers exact test). Also, recurrent pneumoniawas found more frequently among the properdin deficientpatients than among their relatives (in 3 of four patientscompared to 0 of 21 relatives, P=0.0017, Fishers exacttest). Only one case of meningococcal disease hadoccurred. None of the family members could rememberany early deaths or history of severe disease that could berelated to properdin deficiency in relatives living inPakistan or in earlier generations.

Identification of the causative mutation andscreening of the family

Sequencing of the PFC gene on DNA from the index patientIII:1 revealed a novel combined deletion/insertion (del GG/ins CCC) in exon 5. This caused a frameshift from codon 188and resulted in a premature stop codon in codon 217,

Table 1 Number and type of infections reported retrospectively by family members.

NormalN=16

CarriersN=5

Properdin deficient patients N=4

III:1 III:2 III:6 III:13

Otitis media 2×2 a 0 10 8 3 2Sinusitis 2 0 0 0 0 0Pneumonia 1 1 1 3 2 2Meningitis 0 0 0 0 1 0Septicaemia 0 0 3 c 0 1 0Other serious infections 0 2 b 0 0 2 d 1 e

a 2 people reported 2 episodes each.b Herpes zoster and tuberculosis reported in one person.c 3 episodes of septicaemia, suspected clinically and by laboratory findings, though not confirmed by culture.d One episode of subcutaneous abscess, recurrent stomatitis.e Prolonged gastroenteritis.

459Properdin deficiency is associated with recurrent otitis media and pneumonia

Figure 1. The resulting mRNA codes for a protein, which istruncated in TSR3 and lacks TSR4-6 and the C-terminalregion. This truncated protein is not expected to oligomerizedue to the loss of TSR6 [23].

In order to identify carriers of the mutation and proper-din deficient family members, exon 5 was sequenced in

Figure 1 Frameshift mutation in exon 5 of the properdin genePFC. Panel a shows the normal homozygous GenBank sequence.Panel b shows the hemizygous sequence from the properdindeficient patients, the underscored CCC-sequence has replacedthe GG-sequence underscored in panel a. Panel c shows theheterozygous sequence from a female carrier.

relatives of the index patients, Figure 2. This showedthat the mutation was inherited from the grandmother(I:2). Two of her daughters (II:2+6) carried the mutation,and transferred it to their sons (the index patients III:1+2,and their 16-year old cousin III:6). Initially, it was a surprisefor us that III:13 was properdin deficient with the exon 5mutation since his father, who was the son of I:2, did notharbor the mutation. However, this was clarified by aninterview with the family, which revealed that the parentsof III:13 were cousins, and the mother (II:10) was subse-quently shown to be a heterozygous carrier of the mutation.Thus, the deficiency must be inherited from one of thegrandmother's parents.

Identification of a male heterozygous carrier withKlinefelter syndrome

The genetic analysis of the 13-year old boy III:7 showedheterozygosity for the exon 5 mutation, which was unex-pected in a male. Confirmation of the result in a separatesample raised the suspicion of Klinefelter syndrome. This wasconfirmed by chromosome analysis, which revealed thekaryotype 47, XXY. Clinical examination showed bilateralgynecomastia and small firm testes, with a testicular volumeof 5 mL, typical for Klinefelter syndrome. His medical historyrevealed neither increased infection rate nor any previoussevere infections. He had minor developmental and learningdisabilities. The activation of complement through thealternative pathway was 13%, 63% and 39% (normal range30–113%) in three individual samples taken 2–5 monthsapart; the properdin level was 15% and 22% (normal range54–157%) in the first and third sample, respectively.

Discussion

Properdin deficient patients have previously been identifiedmostly by immunological evaluation of patients aftermeningococcal disease or in studies of relatives of thesepatients [7,13,26,28–33]. In the present study properdindeficiency was identified in patients who underwentimmunological evaluation because of recurrent non-menin-gococcal infections. Recurrent infections have not previouslybeen significantly linked to properdin deficiency, even

Figure 2 Pedigree for the Pakistani family. The gray shades indicate deceased family members. Question marks indicate familymembers who were not available for genetic analysis.

460 L. Schejbel et al.

though other infections than Neisseria have been describedin some of the patients [7,13,30,33,34]. A reason for thiscould be that properdin deficiency has been underestimated,because it was not detected by the previous standardscreening assays for complement deficiency such as mea-surement of total hemolytic complement (CH50) [35]. Withthe use of the ELISA based assay Wielisa, the activity of thealternative pathway activity was below 2% in all the patientsin our study. The future use of this assay in immunologicalevaluation of patients with recurrent infections may showwhether the frequency of properdin deficiency has beenunderestimated.

The retrospective study of the infections in the familyshowed an association between properdin deficiency andrecurrent otitis media as well as recurrent pneumonia.Importantly, the questionnaire was completed before thegenetic analysis. Thus the family and their physician wereonly aware of properdin deficiency in the index patients andthe suspicion of properdin deficiency in their cousin (III:6),but unaware of the status of the other family members.Therefore, it seems unlikely that the associations are causedby an increased focus on the properdin deficient familymembers. In previous case reports and family studies ofproperdin deficiency, the focus has been on meningococcaldisease, and much less severe clinical manifestations as otitismedia and pneumonia may easily have been overlooked. Itwould be ideal to confirm this association in a prospectivestudy. This; however,may not be feasible: After identificationof properdin deficiency in a patient, a vaccination programwith quadrivalent polysaccharidemeningococcal vaccine andpolyvalent pneumococcal vaccine program should be per-formed in order to prevent disease. This vaccination programmay also decrease the incidence of otitis media andpneumonia. Also, many properdin deficient patients areidentified in their teens or later, where the incidence of otitismedia is decreased, and therefore, the only way to ascertainthe contribution of properdin deficiency to an increasedsusceptibility to otitis media in these patients is retro-

spectively. Thus, the best way to confirm our findings wouldbe by evaluation of some of the previously described families.An association between MBL-deficiency and recurrent otitismedia in children between 12 and 24 months has also beenreported, indicating that an intact complement activationsystem is important for protection against otitis media in theperiod where the adaptive immunity is not fully developedyet [36], which has also been reported for acute respiratorytract infections [37].

After identification of properdin deficiency in a family it isessential to identify all properdin deficient relatives becauseof the high risk of severe meningococcal disease which has amortality rate of 33% compared to rates of 5–7% inindividuals with terminal complement component deficien-cies and complement-sufficient individuals [5,6,35]. More-over, our study illustrates that even in X-linked recessivediseases as properdin deficiency, it is important to be awareof consanguineous marriages, not because it has anyinfluence on the penetrance of the disease per se, butbecause there may be unexpected carriers of the mutation.In female carriers of properdin deficiency the level ofproperdin in serum may be low or within the normal rangepartly depending on the X-inactivation pattern [38]. There-fore the only way of identification of female carriers is byDNA-based methods. This can be done either by usingmicrosatellites closely linked to the PFC gene [39] or byidentification of the causative mutation in the gene.

In our study we identified a male carrier of the exon 5mutation who was diagnosed with Klinefelter syndrome(47XXY). The prevalence of Klinefelter syndrome is about1.72 in 1000 male births [40]. It is assumed that patients withKlinefelter syndrome are protected against X-linked reces-sive diseases. With respect to properdin deficiency it isnotable that no female carriers of properdin deficiency withmeningococcal disease have been described so far, suggestingthat even women with low levels of properdin in serum areprotected against Neisseria infections. The Klinefelterpatient had decreased properdin levels in two samples.

461Properdin deficiency is associated with recurrent otitis media and pneumonia

Surprisingly, the AP complement activity was low in the firstsample analyzed (13%, normal range 30–113%), although notas decreased as in the properdin deficient patients, who allhad AP activities below 2%. Since the other two samples fromthe Klinefelter patient had normal AP activities we expectedhim to be protected, but had performed the vaccinationprogram. All the female carriers in the family had normal APactivities, but we have not been able to follow these personsover time and therefore do not know whether a fluctuation inAP activity can occur among female carriers of properdindeficiency.

Conclusion: In a Pakistani family, we observed 4 indivi-duals with properdin deficiency. In addition to meningococ-cal disease a striking association with recurrent otitis mediaand pneumonia was revealed. Thus, properdin deficiencyappears to be associated with a larger spectrum of infectionsthan previously appreciated.

Acknowledgments

Excellent technical assistance was performed by EwaSzöjmer. This work was supported by grants from the Novo-Nordisk Research Foundation, Birthe and John MeyerFoundation, Rigshospitalet and the Danish Medical ResearchCouncil.

Appendix A. Supplementary data

Supplementary data associatedwith this article can be found,in the online version, at doi:10.1016/j.clim.2009.02.008.

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