Ficolin-3 Deficiency Is Associated with Disease and an Increased Risk of Systemic Lupus Erythematosus

Abstract

Purpose
Ficolin-3 deficiency is caused by a mutation (+1637delC) in the FCN3 gene. It is a rare condition and has been associated with both infection and autoimmune disease including systemic lupus erythematosus (SLE). Here we investigated if ficolin-3 deficiency is more frequent in patients than in controls and tried to identify a common phenotype among ficolin-3 deficient individuals. Since a significant part of patients identified with ficolin-3 deficiency was diagnosed with SLE, we explored whether the heterozygous state of the FCN3+1637delC variant represents a risk factor in the development of SLE. Further, we examined other possible causes of ficolin-3 deficiency when the FCN3+1637delC is not present.

Methods
A systematic literature search for studies measuring ficolin-3 was carried out. We examined 362 SLE patients and 596 controls for the presence of the variant FCN3+1637delC. We established assays for measurements of ficolin-3 and of auto-antibodies against ficolin-3. We sequenced the coding and non-coding regions of the FCN3 gene in an SLE patient with ficolin-3 deficiency not carrying the +1637delC.

Results
Ficolin-3 deficiency leads to an 8-time increased odds of having a disease (p < 0.05). Three out of nine patients with deficiency had SLE. The heterozygous state of the deficiency variant is not associated with increased risk of developing SLE (p = 0.18).

Conclusion
By systematically reviewing the literature for the described cases of ficolin-3 deficiency, an autoimmune phenotype is emerging. Thirty-three percent of the ficolin-3 deficient patients had SLE. Heterozygosity for the FCN3 gene deletion causing the deficiency does not seem to be associated with the development of SLE.

Full text

ORIGINAL ARTICLE
Ficolin-3 Deficiency Is Associated with Disease and an Increased Risk
of Systemic Lupus Erythematosus
Anne Troldborg
1,2,3
&Rudi Steffensen
4
&Marten Trendelenburg
5
&Thomas Hauser
6
&Kasper G. Winther
2
&
Annette G. Hansen
2
&Kristian Stengaard-Pedersen
3
&Anne Voss
7
&Steffen Thiel
2
Received: 5 March 2019 / Accepted: 7 April 2019
#Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Purpose Ficolin-3 deficiency is caused by a mutation (+1637delC)intheFCN3 gene. It is a rare condition and has been
associated with both infection and autoimmune disease including systemic lupus erythematosus (SLE). Here we investigated
if ficolin-3 deficiency is more frequent in patients than in controls and tried to identify a common phenotype among ficolin-3
deficient individuals. Since a significant part of patients identified with ficolin-3 deficiency was diagnosed with SLE, we
explored whether the heterozygous state of the FCN3+1637delC variant represents a risk factor in the development of SLE.
Further, we examined other possible causes of ficolin-3 deficiency when the FCN3+1637delC is not present.
Methods A systematic literature search for studies measuring ficolin-3 was carried out. We examined 362 SLE patients and 596
controls for the presence of the variant FCN3+1637delC. We established assays for measurements of ficolin-3 and of auto-
antibodies against ficolin-3. We sequenced the coding and non-coding regions of the FCN3 gene in an SLE patient with ficolin-3
deficiency not carrying the +1637delC.
Results Ficolin-3 deficiency leads to an 8-time increased odds of having a disease (p< 0.05). Three out of nine patients with deficiency
had SLE. The heterozygous state of the deficiency variant is not associated with increased risk of developing SLE (p=0.18).
Conclusion By systematically reviewing the literature for the described cases of ficolin-3 deficiency, an autoimmune phenotype
is emerging. Thirty-three percent of the ficolin-3 deficient patients had SLE. Heterozygosity for the FCN3 gene deletion causing
the deficiency does not seem to be associated with the development of SLE.
Keywords SLE .ficolin-3 deficiency .complement .complement deficiency .autoimmunity
Introduction
The plasma protein ficolin-3 (also called H-ficolin and origi-
nally named Hakata antigen) was discovered in 1990 as an
autoantigen when antibodies against the protein were identi-
fied in patients with systemic lupus erythematosus (SLE) [1].
Ficolin-3 is a soluble pattern recognition molecule (PRM) of
the innate immune system. It binds to acetylated molecules,
e.g., as found in acetylated carbohydrate structures or on pro-
teins via its fibrinogen-like recognition domain [2]. The fitting
patterns recognized by ficolin-3 may be present on apoptotic
and necrotic cells, and this gives ficolin-3 the possibility of
acting as an initiator of scavenging actions [3]. When bound to
a surface, ficolin-3 can activate the complementsystem via the
lectin pathway (LP), giving rise to both anti-microbial defense
and homeostatic balance. To enable this activation, ficolin-3
relies on activation of attached enzymes, i.e., so-called
Mannose-binding lectin (MBL)-associated serine proteases
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10875-019-00627-2) contains supplementary
material, which is available to authorized users.
*Anne Troldborg
annetrol@rm.dk
1
Department of Rheumatology, Aarhus University Hospital, Palle
Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark
2
Department of Biomedicine, Aarhus University, Aarhus, Denmark
3
Department of Clinical Medicine, Aarhus University,
Aarhus, Denmark
4
Department of Clinical Immunology, Aalborg University Hospital,
Aalborg, Denmark
5
Division of Internal Medicine and Clinical Immunology lab,
Department of Biomedicine, University Hospital Basel, University of
Basel, Basel, Switzerland
6
IZZ Immunologie-Zentrum Zürich, Zürich, Switzerland
7
Department of Rheumatology, Odense University Hospital,
Odense, Denmark
Journal of Clinical Immunology
https://doi.org/10.1007/s10875-019-00627-2

(MASPs) [4]. Ficolin-3 is the most abundant of the five PRMs
of the LP (as compared with ficolin-2, ficolin-1, MBL, and
collectin-LK).
In our previous investigations, we have consistently ob-
served high levels of ficolin-3 in patients with SLE [5,6].
This is in line with investigations by other research groups
[1,7,8]. However, when examining a large SLE cohort of
424 patients, we also identified two ficolin-3 deficient indi-
viduals [6]. One of the patients carried the only known
mutation-causing ficolin-3 deficiency; the other patient
(patient-X) did not.
Ficolin-3 is encoded by the FCN3 gene. A functional gene
is only present in primates whereas, e.g., the rodent FCN3
orthologue is a pseudogene [9,10]. Among the known poly-
morphisms of FCN3, only one leads to complete deficiency in
variant homozygotes. A frameshift mutation causing ficolin-3
deficiency is located on exon 5 of the FCN3 gene
(+1637delC, rs532781899) [11]. It has a gene frequency of
0.01, and thus, homozygosity is expected in 1 out of 10,000
individuals [12,13]. Before our findings in a cohort of SLE
patients [6], only males with ficolin-3 deficiency had been
identified [14].
The PRMs of the LP provide defense against a large array
of pathogens and are primarily mediated by recognition of
highly conserved pathogen-associated molecular patterns,
i.e., repetitive sugar arrays on the surface of microorganisms
that are rare or non-existent on mammalian cells [15].
Deficiencies of the PRMs are therefore hypothesized to be
associated with serious infections by several research groups
[16,17]. Ficolin-3 deficiency has only been described in a
limited number of humans, and the alleged infectious pheno-
type is controversial [14]. Secondary ficolin-3 deficiency
caused by autoantibodies against the protein has been de-
scribed in SLE patients and has been suggested as a biomarker
for disease activity and glomerulonephritis [1,18,19].
SLE is a chronic autoimmune disease. The disease course
is unpredictable and characterized by periods of remission
followed by periods of ongoing disease activity (flares).
Defects in the clearance of apoptotic cells leading to the re-
lease of intracellular autoantigens resulting in the induction of
autoantibody production have been proposed as a key patho-
genic mechanism of SLE [20,21]. In agreement with this,
proteins that are implicated in the stringently orchestrated
clearance of dying cells are likely to play a role in host pro-
tection against SLE.
The objectives of the current study were to investigate if
ficolin-3 deficiency would be more common in patients than
in controls by reviewing published literature on ficolin-3 and
identifying the number of patients and controls that had been
investigated. We aimed to identify a common phenotype
among ficolin-3 deficient individuals. Further, since a signif-
icant part of patients identified with ficolin-3 deficiency were
diagnosed with SLE, we explored whether the heterozygous
state (C/del) of rs532781899 would represent a risk factor in
the development of SLE. Lastly, we examined an SLE patient,
who is ficolin-3 deficient, but not carrying the +1637delC
mutation for other potential causes leading to ficolin-3 defi-
ciency. Further, we describe the first female patient with
ficolin-3 deficiency.
Methods
Study Populations
SLE patients followed at the out-patient clinic of the
Department of Rheumatology,
Aarhus University Hospital, Denmark (n=169), the
Rheumatology out-patient clinic of Odense University
Hospital, Denmark (n= 203), and SLE patients from the rheu-
matology out-patient clinic of Basel, Switzerland, (n= 52)
have previously been described [6]. The Danish cohort is a
cross-sectional cohort and represents patients in full remission
as well as patients with active disease. All patients fulfilled the
1997 revised ACR classification criteria for SLE [22].
Controls (n= 596) used in the study were included with in-
formed consent at the blood bank at Aalborg University
Hospital, Denmark.
Patient-X
The patient is a male SLE patient (from our Danish SLE cohort)
diagnosed at the age of 27 with classical SLE symptoms (ar-
thritis, malar rash, lupus nephritis, positive anti-dsDNA, anti-
nuclear antibodies, low complement). In the 8-year course of
disease, he has had three severe nephritis flares. He currently
has ongoing proteinuria of more than 5 g/day and is treated with
prednisolone (10 mg/day), mycophenolate-mofetil (3 g/day),
and hydroxychloroquine (400 mg/day). The patient was admit-
ted with biopsy-verified vasculitis of the colon in 2015 treated
with high dose intravenous prednisolone. He has not had doc-
umented recurrent infectious episodes.
First Female Patient with Ficolin-3 Deficiency
The patient is a Swiss SLE patient (from our own SLE cohort)
diagnosed at the age of 18 with classical SLE symptoms (ar-
thritis, pleuritis, malar rash, lupus nephritis, positive anti-
dsDNA, anti-nuclear antibodies, low complement, lupus
headache, Coombs-positive anemia, lymphopenia, high anti-
C1q). One year after diagnosis, she presented with meningo-
coccal meningitis (type non-B/C) in cerebrospinal fluid, and
in peripheral blood (culture), consecutively, she developed
septic shock with septic cardiomyopathy, acute renal failure,
disseminated intravascular coagulation, and respiratory fail-
ure. At the time, she was under mild immunosuppression with
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prednisone (10 mg/day) and azathioprine (50 mg/day). She
was successfully treated with ceftriaxone. Four years after
her SLE diagnosis, she presented with severe pneumonia
(without identification of a pathogen), and 5 years after diag-
nosis with acute Escherichia coli cystitis with pyelonephritis.
In the same year, she had a renal flare and was started on
mycophenolate-mofetil. The mother of this patient was also
diagnosed with SLE and was found to be heterozygous for the
FCN3 gene deletion [6].
Literature Review Search
Articles were identified by systematic literature searches in
MEDLINE, Embase, Web of science, and Scopus covering
the period from 1980 to January 1, 2018. The following search
terms were used and adjusted based on the search engine used:
BH-ficolin^,BFicolin 3^,BFCN3 gene^,BFicolin^,
BComplement Pathway, Mannose-Binding Lectin^.
Inclusion criteria were clinical studies on humans where
ficolin-3 concentrations had been measured in a patient group
and/or control group, or studies where genetic testing for
ficolin-3 deficiency had been performed.
Exclusion criteria were studies in which methods were not
described sufficiently, data were not available for evaluation,
studies in which neither ficolin-3 plasma or serum concentra-
tions nor genetic data were available, or review articles not
containing original data (Supplementary Fig. 1outlines the
screening and selection process of the literature search).
After removing duplicates, the remaining articles were
screened by title and abstract based on the exclusion criteria,
which left 64 articles for full-text assessment. Sixty-four arti-
cles were screened independently and evaluated for inclusion
and exclusion criteria. Ten of these articles were excluded due
to missing information on measured plasma concentrations. In
the remaining 54 articles, the number of patients and controls
examined for ficolin-3 were then added up. Results from the
current article and the article in which our two ficolin-3 defi-
cient SLE patients were identified [6] were added to the group
of articles ending up with a total of 56 articles.
PCR Analysis for FCN3+1637delC Mutation
DNA from SLE patients and controls were extracted from
peripheral blood mononuclear cells (PBMCs) using the
Maxwell16 Blood DNA kit on the Maxwell16 Instrument
(Promega).
To cover the frameshift mutation FCN3+1637delC
(rs532781899) [12], a fragment of 366 bp of FCN3 exon 5
was amplified by PCR using the following primers: 5′-
ggccaagatcctccccaca-3′and 5′-tctggtgggttctggctcc-3′.PCR
amplifications were carried out in 50 μl volumes containing
~ 50 ng genomic DNA, 0.5 mM of each primer, 1× PCR
buffer II, 2.5 mM MgCl2, 0.2 mM dNTP, and 0.75 units of
AmpliTaq DNA polymerase (Invitrogen Life Technologies).
The PCR reactions were performed at; 5 m94 °C, 35 cycles
(30 s94 °C, 30 s62 °C, 30 s72 °C), 5 m72 °C.
After a cleaning step (FlashGel Recovery System, Lonza,
Inc.), the fragment was sequenced in both directions using the
ABI BigDye cycle sequencing terminator kit, V 1.1 (Applied
Biosystems). PCR amplifications were performed in 20 μl
volumes at: 1 m96 °C, 25 cycles (10 s96 °C, 5 s50 °C,
4 m60 °C). PCR products were purified (BigDye
XTerminator, Applied Biosystems) and sequences analyzed
(ABI Prism 3500 Genetic Analyzer, Applied Biosystems).
CLC main workbench software was used for alignment of
resulting DNA sequence.
FCN3 Gene Sequencing
A total of 6510 bases of the FCN3 gene (covering all 8 exons
of the gene) were sequenced covering 1350 base pairs (bp)
upstream of exon 1, exon 1, intron 1, exon 2, intron 2, exon 3,
intron 3, exon 4, intron 4, exon 5, and 300 bp upstream of
intron 5, 482 bp downstream of exon 6, exon 6, intron 6, exon
7, and 271 bp upstream of intron 7, 308 bp downstream of
exon 8, besides 1485 bp upstream of the 3’UTR part. The
sequence was aligned to the NG_016279 sequence from the
NCBI nucleotide database. The sequencing was performed as
a resequencing service at Eurofins Genomics GmbH Freiburg,
Germany.
Ficolin-3 Assay
Microtitre plates (Nunc, #437958 or # 43791) were incubated
with acetylated bovine serum albumin (Ac-BSA) (Sigma
#B2518) 10 μg/ml coating buffer (0.1 M sodium bicarbonate,
pH 9.6 with 0.09% (w/v) sodium azide, and Ampliqon
#AMPQ44048.1000) and incubated overnight at room tem-
perature (RT). Residual binding sites were blocked by incu-
bation for 1 hour (h) with 200 μl of 1 mg human serum albu-
min (HSA)/ml TBS (10 mM Tris, 145 mM NaCl, pH 7.4) per
well, and plates were washed three times with TBS/TW (TBS
with 0.05% Tween 20). Standard, quality controls, and sam-
ples were then added to the plate in duplicates. The standard
curve was obtained by diluting the standard (pool of human
plasma with a known concentration of 20 μg/ml ficolin-3) 1/
10 and further seven times twofold dilutions with TBS/Tween/
Ca
2+
/HSA (TBS/TW, 5 mM CaCl
2
, and 1 mg/ml HSA). The
concentration of ficolin-3 in the standard is based on compar-
isons of the signal obtained in dilutions of standard with that
obtained in dilutions of purified plasma ficolin-3. Samples and
quality controls were diluted 1/50 in TBS/Tween/Ca
2+
/HSA.
After overnight incubation at 4 °C and three times wash in
TBS/Tween/Ca
2+
biotinylated mouse anti-human-ficolin-3,
clone 4H5 (conc. 0.34 μg/ml, Hycult biotechnology
#HM2089b) diluted 1/1000 in TBS/Tween/Ca
2+
were added
JClinImmunol

to each well at 100 μl/well and incubated for 2 h at RT. The
subsequent steps followed our standard procedures for time-
resolved immunofluorometric assay (TRIFMA) as previously
described [6], i.e., incubation with europium-labeled
streptavidin followed by wash and detection of the signal by
time-resolved fluorometry.
Purification of Ficolin-3 from Serum
Ficolin-3 was purified from human serum following a previ-
ously described procedure [23]. Serum was precipitated with
polyethylene glycol (PEG) in two steps each followed by cen-
trifugation, and at 4 °C, the supernatant was passed through a
10 ml HSA-sepharose (HSA coupled to CNBr-activated se-
pharose beads) column (this removes most L-ficolin), and the
effluent was loaded onto a 20 ml-acetylated HSA-sepharose
column to bind ficolin-3. The column was washed with 80 ml
of TBS/tw/Ca
2+
. Ficolin-3 was subsequently eluted from the
beads with 1 M sodium acetate, pH 7.5, 1 ml/fraction.
Fractions with ficolin-3 were pooled and dialyzed against
50 mM sodium acetate, 0.01% Tween-20, and pH 8.3. After
dialysis, the preparation was centrifuged 9000gfor 10 min.
The ficolin-3 in the supernatant was purified further on an ion
exchange column MonoQ 5/50 GL (GE Healthcare), using a
gradient from 50 mM NaCl to 350 mM NaCl over 30 ml. The
purity of the protein was tested by SDS-PAGE protein stain
(Supplementary Fig. 2).
Assay Measuring Auto-antibodies Against Ficolin-3
Microtitre plates (Nunc, Roskilde, Denmark; # 437958 or #
43791) were incubated with purified ficolin-3 at 0.5 μg/ml
phosphate buffered saline (PBS) overnight at RT. Residual
binding sites were blocked with 1 mg of HSA per ml of
TBS. After washing with TBS/TW, 100 μl of test samples
and three quality controls (plasma chosen to reflect a wide
distribution of concentrations) were added to the plate diluted
1/10 in sample buffer (10 mM Tris/base, 145 mM NaCl,
10 mM EDTA, 0.05% Tween-20, 1 mg HSA/ml, heat-
aggregated human immunoglobulin (Ig)G 100 mg/ml,
pH 7.4). All samples were added in duplicates using a 1/10
dilution. A standard curve was constructed from a pool of four
SLE EDTA plasma samples, initially diluted tenfold followed
by serial threefold dilutions. After incubation overnight at
4 °C, the wells were washed with TBS/TWand incubated with
biotin-rabbit-anti-human-kappa/lambda (Dako #A0194 and
A0191) at 1/5000 in TBS/TW for 2 h at RT. After washing
with TBS/TW, the wells were incubated with europium-
labeled streptavidin (Perkin Elmer, USA; #1244-360) 1/1000
in TBS/TW, 25 μM EDTA for 1 h at RT. After washing,
quantification of europium was performed by adding 200 ml
of enhancement solution (Ampliqon laboratory reagents
#Q99800; Ampliqon, Denmark) per well releasing and
encapsulating the bound europium, and the fluorescence was
read as time-resolved fluorometry on a Victor 5 from Perkin
Elmer.
As a control, plates were also coated with human IgG to be
able to adjust for the difference in background signal between
SLE patients and controls. No significant binding was ob-
served for neither SLE patient sera nor for controls, and no
difference in background signal was observed between pa-
tients and controls.
Western Blot
We tested whether ficolin-3 protein from patient-X could be
detected by Western blot and compared with the signals seen
in other SLE patient samples. Serum samples from six SLE
patients with known ficolin-3 serum concentrations (one de-
ficient, two with high concentrations, one with medium con-
centrations and two with low concentration), based on mea-
surements in TRIFMA assays as described above, were added
to SDS PAGE sample buffer (30 mM Tris-HCL, 10% (v/V)
glycerol, 8 M urea, 3% (w/v)SDS,0.1%(W/v) bromophenol
blue, pH 8.9). TBS was added to reach the desired sample
volume (30–45 μl). Dithiothreitol (DDT) was added to reach
60 mM in the samples to be reduced, followed by heating and
addition of iodoacetic acid. Proteins were separated using an
18 well 4–15% gradient gel (Bio-Rad, Criterion TGX gels #
567-1084). Following electrophoresis, the proteins were blot-
ted onto nitrocellulose membranes (Bio-Rad #170-4159). The
membranes were then blocked by incubation for 30 min at RT
in TBS with 0.1% Tween (v/v), washed, and developed with
goat-anti-human-ficolin-3 (RD#F2367) at 0.5 mg/ml) in pri-
mary buffer (TBS, 1 mM EDTA, pH 7.4, with 1 mg HSA
(CSL Behring #109697) and 100 μg human IgG (CSL
Behring #007815) per ml). The membrane was subsequently
washed and incubated with HRP-conjugated rabbit-anti-goat
IgG antibody (DAKO #P0449) diluted 1/3000 in secondary
buffer (TBS/Tween, 100 μg human IgG/ml, 1 mM EDTA,
pH 7.4). After washing, the blot was developed with
SuperSignal West Dura extended duration substrate (Pierce),
and emission recorded by a charge-coupled device camera.
Ethics
The project was performed according to the Helsinki
Declaration. The Danish Data Protection Agency and the
Central Denmark Region Committees on Health Research.
Ethics approved the study conducted in Aarhus (#1-10-72-
214-13). The Southern Denmark Region Committees on
Health Research Ethics approved the inclusion of patients in
Odense (#2010 0015). For inclusion of the Swiss SLE popula-
tion, the Ethical committee of Northwest and Central
Switzerland approved the project (EKNZ.Ref. no. EK 262/06).
JClinImmunol

Table 1 Patients with ficolin-3 deficiency described in the literature
Clinical presentation Age Gender (M/F) Ficolin-3 concentration
μg/ml (plasma or serum)
Homozygosity for the
FCN3+1637delC
Autoantibodies
against ficolin-3
Reference Dominant
phenotype
Recurrent pulmonary infections, brain abscesses,
and recurrent warts on fingers; bronchiectasis
and pulmonary fibrosis
32 M < 0.1 Yes NA [12] Autoimmune
SLE no other clinical data available NA NA < 1.1 No No [7] Autoimmune
Invasive necrotizing enterocolitis and repeated
skin infections with staphylococci; no other
infectious diseases during 4-year follow-up
29 weeks GA M < 0.1 Yes NA [24] Infectious
Fatal necrotizing enterocolitis 28 weeks GA M 0.8 NA NA [24] Infectious
Perinatal S. agalactiae infection; microcephaly,
growth and mental retardation; ADHD; no
severe infections during 8-year follow-up
35 weeks GA M < 0.1 Yes NA [25] Infectious
Membranous nephropathy; nephrotic syndrome;
EBV infection; no other recurrent infections
50 M < 0.1 Yes NA [14] Autoimmune
Congenital heart disease; pneumonia; no severe
infections during follow-up after cardiac
surgery
11 months M < 0.1 Yes NA [14] Infectious
Healthy individual (control person) no clinical
data available
NA NA < 0.1 Yes NA [26]NA
Healthy individual (control person) no clinical
data available
NA NA < 0.1 Yes NA [26]NA
SLE (arthritis, positive ANA, anti-ds-DNA,
glomerulonephritis, low C3 and C4).
Meningococcal sepsis. 2nd severe lung
infections.
18 F < 0.1 Yes NA [6] Autoimmune
SLE (malar rash, arthritis, glomerulonephritis,
ANA positive, anti-ds-DNA positive,
low complement C3 and C4)
27 M 0.3 No No [6] Autoimmune
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Results
Ficolin-3 Deficiency
Based on our literature search, a total of 10 individuals with
ficolin-3 deficiency are now described in the literature, nine
males and one female. All individuals with described ficolin-3
deficiency where evaluated for disease phenotype, age, gen-
der, ficolin-3 concentration, genetic data, and autoantibody
status against the ficolin-3 protein where this information
was available (Table 1).
Based on all published data on ficolin-3, we observed that a
total of 9294 patients and 8227 controls had been evaluated
for either ficolin-3 concentration and/or ficolin-3 deficiency
by genetic testing (Supplementary Table 1). These numbers
are based on the addition of the total number of patients and
the total number of controls reported in the included articles of
the literature search. Among these two groups, nine patients
and only one control were deficient for the FCN3del1637,
yielding an odds ratio for having a disease phenotype when
being ficolin-3 deficient of 8.0 (95% CI 1.01–63.0, p<0.05)
(Table 2).
FCN3 Mutation in SLE
Since three of the nine identified patients with ficolin-3 deficien-
cy were SLE patients, we examined (in our own cohorts) whether
being a heterozygous carrier of the FCN3 + del1637C would be
a risk factor for the development of SLE. SLE patients (n=362)
and controls (n= 596) were assessed for the mutation. In SLE
patients, 2.8% carried the mutation, and in controls, this was true
for 1.5%. This difference was not significant (p= 0.18) (Fig. 1).
Investigations of the Cause of Ficolin-3 Deficiency
in Patient-X
Sixteen randomly chosen SLE patients from our SLE cohort
with different serum concentrations of ficolin-3 and our ficolin-
3 deficient patient without the known mutation-causing ficolin-
3 deficiency (patient-X), and 18 randomly chosen controls were
examined for auto-antibodies against ficolin-3. No significant
difference was observed between controls and SLE patients
(Fig. 2a),andnocorrelationwasobserved between ficolin-3
plasma concentration and concentration of auto-antibodies
against ficolin-3 (Fig. 2b).Onepatienthadveryhighlevelsof
antibodies against ficolin-3; however, the patient had normal
plasma levels of ficolin-3 (38 μg/ml). Patient-X did not have
higher concentrations of autoantibodies against ficolin-3 than
the controls (open circle Fig. 2a).
To examine whether ficolin-3 protein was present in
patient-X in a form not detectable in our immunoassay, we
investigated patient serum by Western blotting. Ficolin-3 is
normally detected at 34 kDa under reducing conditions
(Fig. 3a) and as several high oligomeric bands above
250 kDa under non-reducing conditions (Fig. 3b). No
Fig. 1 Number of carriers of the
heterozygous mutation FCN3+
1637delC in Danish SLE patients
and controls. In the SLE patients,
2.8% (10 / 362) were heterozy-
gous carriers of the FCN3+
1637delC, the same was true for
1.5% (9/596) of the controls. P
value reflects χ
2
for the difference
between the two groups
Table 2 Cumulated number of patients and controls in the published
literature where ficolin-3 has been measured based on a systematic liter-
ature review. AnOR of havingdisease when being ficolin-3 deficient was
calculated based on the number of ficolin-3 individuals in each group
(patients and controls)
Ficolin-3 measured Patients (p) Controls (c) Deficient (p/c)
Total 9.294 8.227 9/1
Odds for disease when being ficolin-3 deficient: OR 8.0 (95% CI 1.01–
63.0, p<0.05)
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ficolin-3 was detectablein patient-X (lane 1 Fig. 3a (reducing)
and lane 1 Fig. 3b (non-reducing)).
For patient-X, genetic sequencing of all exons [1–8]and
flanking sequences of the FCN3 gene was carried out to detect
potential new mutations to account for the observed deficien-
cy. No such mutations were discovered. Only a heterozygous
mutation located upstream of exon1 in the promoter region
was observed (Supplementary Fig. 3c).
Fig. 3 Test for ficolin-3 using Western blotting of samples from the SLE
deficient patient not carrying FCN3 1637delC (patient-X). Proteins were
separated on a 4–15% gel and blotted, and the blot developed with goat-
anti-ficolin-3. aFicolin-3 is detected at 34 k Da under reducing conditions
(lanes 2, 3, 4–6). No ficolin-3 was present in patient-X (lane 1). bFicolin-
3 is detected as several oligomeric bands above 250 kDa under non-
reducing conditions. No ficolin-3 was present in patient-X (lane 1, a +
b). Lanes 2–3and5–7 on each blot represents serum from SLE patients
with high (lane 2–3), medium (lane 5), and low [6,7] concentrations in
serum of ficolin-3
Fig. 2 Auto-antibodies against ficolin-3. aAuto-antibodies against
ficolin-3 in SLE patients and controls. Open circle represents patient-X
with ficolin-3 deficiency not carrying the known mutation causing
ficolin-3 deficiency (FCN3 + 1637delC). Auto-antibodies against
ficolin-3 did not explain the observed deficiency. bCorrelation between
ficolin-3 plasma concentrations and auto-antibodies against ficolin-3
JClinImmunol

Discussion
By systematically reviewing the literature, we identified 10
individuals with ficolin-3 deficiency. Eight carried the known
FCN3del1637 mutation. Three of the ten described cases had
a diagnosis of SLE. The heterozygous state of the
FCN3del1637 mutation, which leads to a 50 % reduction in
ficolin-3 plasma concentration was not associated with the
development of SLE.
Our immediate hypothesis was, that the discovery of
ficolin-3 deficiency in diseased individuals, could be due to
diseased populations being studied more rigorously than con-
trols. However, when evaluating the literature, we found that
approximately the same number of diseased individuals and
healthy individual hasbeen investigated since the discovery of
ficolin-3. Based on the currently available literature, this im-
plies that the deficient state is a risk factor for disease and not
just a selection bias. The clinical consequences of ficolin-3
deficiency are still unclear and definite conclusions cannot
be drawn based on 10 individuals. However, a pattern of in-
fection in the young cases and an autoimmune phenotype in
the adult cases, does seem to be emerging.
Michalski et al. reported, that heterozygosity for the FCN3
gene deletion does not seem to have major clinical importance
in neonates [25]. This also appears to be the case for SLE, i.e.,
we did not find a significantly higher number of heterozygous
carriers among SLE patients. In concurrence with previous re-
ports, ficolin-3 concentrations in the heterozygous patients were
on average half of the expected normal concentration [6,12].
In patient-X, we were not able to detect a genetic explana-
tion for the observed deficiency. We did find a heterozygous
mutation upstream of exon1. It is, however, unlikely to be the
explanation of the deficiency in patient-X, since the mutation
was heterozygous. Autoantibodies against ficolin-3 could be
an explanation for a pseudo deficient state. This phenomenon
is known from complement C1q, where autoantibodies
against the protein are strongly associated with protein levels
and disease activity [27,28]. Autoantibodies against ficolin-3
were recently reported in as much as 37% of the SLE popu-
lation investigated [18]. We did, however, not observe higher
levels in patient-X, and although only a small number of pa-
tients and controls were examined in the present study (SLE
n= 16, controls n= 16), we did not (on average) see any
difference in antibodies against ficolin-3 between SLE pa-
tients and controls, nor did we see a correlation between
ficolin-3 levels and antibodies against ficolin-3. The deficien-
cy could be caused by epigenetic regulation. Epigenetic regu-
lation of the FCN3 gene has been described in relation to high
ficolin-3 levels in leprosy [29]. We did, however, not have
liver tissue available from patient-X, which would be neces-
sary to investigate this hypothesis further.
In the original studies on antibodies against ficolin-3 by
Inaba et al, 283 SLE patients and 398 patients with other
autoimmune diseases were examined [1]. In the SLE cohort,
15 patients with unmeasurable amounts officolin-3 were iden-
tified, 3 of who were also positive for antibodies against
ficolin-3. In the group of other autoimmune diseases, two
patients were identified as ficolin-3 deficient without detect-
able antibodies against ficolin-3, one with chronic glomerulo-
nephritis and one with primary biliary cirrhosis. The study
was carried out prior to the purification of ficolin-3, and the
authors proposed the deficient state to be caused by autoanti-
bodies against ficolin-3 although they did not detect antibod-
ies against ficolin-3 in most of the patients.
Based on the DNA deposits gathered at the Broad Institute
[13], 26 people homozygous for the FCN3 del1637 mutation
have been detected resulting in an allele frequency of 0.017.
Clinical data on these individuals are not available.
The question still remains, whether ficolin-3 deficiency is
the cause of disease or acts as a disease modifier. In relation
to infections, it has been proposed that the crucial role of
ficolin-3 may be to control normal (commensal) flora that
can cause opportunistic infections rather than to protect from
obligatory pathogens [14]. With regard to autoimmunity, it
has been demonstrated, that ficolin-3 binds to apoptotic cells
and activates the complement system [3,30]. It is well
established, that clearance defects are strongly associated
with autoimmune diseases [20], and it is striking to see that
among the diseased adults with ficolin-3 deficiency SLE,
glomerulonephritis, and lung fibrosis are the clinical
presentations.
We speculate that ficolin-3 deficiency, like C1q deficiency,
could increase the risk of serious infections especially in early
childhood; whereas, the deficient state in adulthood is a po-
tential risk factor for autoimmune diseases like SLE, in which
clearance defects seem to play an important role.
Acknowledgements We thank the Swiss Systemic Lupus Erythematosus
Cohort Study (SSCS) for collaboration in the project. We are grateful to
all patients participating in the project.
Authors Contribution AT, ST, and MT designed the study. AT performed
the literature search. AT, AH, and KG performed the laboratory experi-
ments. RS performed the genetic analysis. AT was in charge of collecting
blood samples and handling the blood samples after they were drawn; AT,
AV, and KS handled patient inclusion and clinical assessments. ST devel-
oped the assays used in the project and supervised laboratory procedures.
AT, ST, and KS wrote the manuscript, and all authors participated in the
editing of the article.
Funding Information The authors would like to acknowledge the Danish
Rheumatism association, Aase and Ejnar Danielsens fond, Lundbeck
Foundation, and the Danish National Research Foundation for financial
support.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
JClinImmunol

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