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Single nucleotide polymorphisms in genes encoding toll-likereceptors 7, 8 and 9 in Danish patients with systemic lupuserythematosus
C. Enevold • C. H. Nielsen • R. S. Jacobsen •
M. L. F. Hermansen • D. Molbo • K. Avlund •
K. Bendtzen • S. Jacobsen
Received: 13 November 2013 / Accepted: 3 June 2014
� Springer Science+Business Media Dordrecht 2014
Abstract Several studies indicate a role for toll-like
receptors (TLRs) in the pathogenesis of systemic lupus
erythematosus (SLE). We aimed to investigate the risk of
SLE and typical clinical and serological manifestations of
SLE potentially conferred by selected single nucleotide
polymorphisms (SNPs) of genes encoding TLR7, TLR8,
and TLR9. Using a multiplexed bead-based assay, we
analyzed eight SNPs in a cohort of 142 Danish SLE
patients and a gender-matched control cohort comprising
443 individuals. Our results showed an association between
the rs3853839 polymorphism of TLR7 and SLE (G vs. C,
P = 0.008, OR 1.60, 95 % CI 1.12–2.27 in females;
P = 0.02, OR 4.50, 95 % CI 1.18–16.7 in males) con-
firming recent findings in other populations. Additionally,
an association between the rs3764879 polymorphism of
TLR8 and SLE (G vs. C, P \ 0.05, OR 1.36, 95 % CI
0.99–1.86 in females; P = 0.06, OR 4.00, 95 % CI
0.90–17.3 in males) was found. None of the other inves-
tigated SNPs were associated with SLE but several SNPs
were associated with clinical and serological manifesta-
tions. In summary, a previously shown association between
the rs3853839 SNP of TLR7 and SLE in Asian patients was
also found in Danish patients. Together with the associa-
tion of several other SNPs of TLR8 and TLR9 with various
clinical and serological manifestations of SLE these find-
ings corroborate the pathogenic significance of TLRs in
SLE.
Keywords Systemic lupus erythematosus � Toll-like
receptors � Single-nucleotide polymorphism � Nephritis
Introduction
Systemic lupus erythematosus (SLE) is a systemic auto-
immune disease characterized by manifestations from
several organs dominated by skin, joint and renal mani-
festations, but also from serosal and mucosal surfaces, the
hematological system, and the nervous system [1], [2].
Nephritis stands out from these manifestations by its
prevalence and severity [3]. Furthermore, SLE is charac-
terized by a plethora of autoantibodies [4], namely against
nuclear antigens with practically all patients having anti-
nuclear antibodies (ANAs) at some point. Such antinuclear
antibodies include antibodies against dsDNA, Smith
C. Enevold (&) � C. H. Nielsen � R. S. Jacobsen �M. L. F. Hermansen � K. Bendtzen � S. Jacobsen
Institute for Inflammation Research, Department of Infectious
Medicine and Rheumatology, Rigshospitalet, Copenhagen,
Denmark
e-mail: [email protected]
S. Jacobsen
e-mail: [email protected]
D. Molbo � K. Avlund
Section of Social Medicine, Department of Public Health,
University of Copenhagen, Copenhagen, Denmark
K. Avlund
Center for Healthy Aging, University of Copenhagen,
Copenhagen, Denmark
K. Avlund
Danish Aging Research Center, University of Aarhus, Aarhus,
Denmark
K. Avlund
Danish Aging Research Center, University of Southern
Denmark, Odense, Denmark
K. Avlund
Danish Aging Research Center, University of Copenhagen,
Copenhagen, Denmark
123
Mol Biol Rep
DOI 10.1007/s11033-014-3447-4
antigen and U1-ribonucleoprotein (RNP) and may be
present several years before onset of clinical manifestations
[5].
The specific causal links between these autoantibodies
and clinical manifestations remain to be elucidated. In both
murine and human lupus nephritis, however, deposition of
immune complexes containing chromatin along the glo-
merular and skin basement membranes has been demon-
strated, suggesting a common pathogenetic pathway for
lupus related nephritis and exanthema [6].
The production of autoantibodies is the result of an
expansion of autoreactive B cells assisted by autoreactive T
cells that have escaped central and peripheral tolerance
mechanisms. Breakage of peripheral tolerance in SLE may
be associated with deficient function of regulatory T cells
[7], which may, in turn, relate to aberrations in regulatory
B cells [8]. Defective clearance of nuclear constituents is
hypothesized to be of central importance in the pathogen-
esis of SLE [9] by increasing the amount of nuclear con-
stituents available for presentation to the immune system,
either in free soluble form (as seen for free circulating
DNA [10]) or via nucleotide-loaded microparticles derived
from apoptotic cells [11]. In either form, pattern recogni-
tion receptors such as the toll-like receptors (TLR) are
essential for nucleotide recognition and signal induction in
antigen-presenting cells. TLR7, TLR8, and TLR9 are able
to sense single stranded (ss) RNA and unmethylated CpG-
containing DNA, respectively, and several studies point to
the emerging role of these TLRs in the pathogenesis of
SLE [12].
In male BXSB mice, a segmental duplication containing
TLR7 enhances autoreactive B cell responses to RNA-
related antigens, leading to lupus-like disease [13], [14].
This association between SLE and TLR7 was recently
corroborated by a large genetic study including multiple
populations of Asian descent [15]. In that study, the iden-
tified risk allele of TLR7-rs3853839 also conferred ele-
vated expression of TLR7 in vivo, accompanied by a more
pronounced interferon signature in peripheral blood
mononuclear cells.
Upon activation by their cognate ligands, TLR7, TLR8,
and TLR9 are capable of inducing production of Type I
IFNs, including IFN-alpha [16]. Type I IFNs normally
serve an important function in the defense against viral
infection but in SLE elevated levels of serum Type I IFN is
frequently observed and is of importance to several aspects
of the pathology and etiology of SLE (reviewed by [16])
representing a possible direct link between TLR and SLE.
Moreover, interferon regulatory factor 5 (IRF5) is a
central mediator of TLR7- and TLR8-signaling that pro-
motes the production of type I IFNs, including IFN-alpha,
as well as other inflammatory cytokines. Genetic variation
in the IRF5-encoding gene has been shown to be strongly
associated with SLE, supporting a role for this particular
pathway in the development of the disease [17, 18].
Interestingly, Epstein-Barr Virus (EBV), which has
been associated with SLE and other autoimmune dis-
eases, promotes IFN-alpha production through TLR-sig-
naling and affects regulation of TLR7, TLR9, and IRF5
[19–21].
A role of TLR8 in SLE pathogenesis has not been
clearly defined, but plasmacytoid dendritic cells are acti-
vated by co-stimulation through TLR7 and TLR8 by self-
RNA [22, 23]. Only few single nucleotide polymorphism
(SNP) analyses have been performed on TLR8, and most of
these have not revealed any association with SLE [24].
However, a large study uncovered associations to SNPs in
TLR8 across several ethnical groups [17].
There is only limited and conflicting evidence for an
association between TLR9 and risk of SLE [25]. However,
both TLR7 and TLR9 mRNA are highly expressed in
peripheral blood mononuclear cells of SLE patients and the
expression correlates with disease activity [26]. Moreover,
TLR7, TLR8, and TLR9 are highly expressed in murine
and human lupus nephritis [27]. TLR7 and TLR9 may have
opposing roles as to the development of renal disease in
SLE mouse models, and lupus-related renal disease is
exacerbated in TLR9-deficient mice [28, 29]. In this regard,
it is of interest that TLR9 seems to play a pivotal role in the
induction of tolerance towards apoptotic cell material
through IL-10 producing B cells [30].
Given the roles of TLR7, TLR8, and TLR9 in immune
activation and, potentially, in SLE pathogenesis, we
investigated the relationship between SLE and selected
manifestations of SLE with selected SNPs of TLR7, TLR8,
and TLR9 in a Danish population of SLE patients and
gender-matched controls.
Materials and methods
Patients and controls
Consecutive, unrelated, patients diagnosed with SLE
according to classification criteria by the American College
of Rheumatology (ACR) [31, 32] were included from the
in- and out-patient clinics of a tertiary referral rheumatol-
ogy center with special interest in SLE. Prevalent in- and
out patients aged [18 years were eligible for inclusion.
The included patients, which have been subjected to pre-
vious studies [33], comprised a cohort of 142 patients (132
women, 10 men) with mean disease duration of 15 years
(Table 1). Because of the low number of male participants
and because of the inherent differences between hemi- and
homozygous carriage of X chromosome-bound alleles,
only the results for women were included in Tables 2, 3
Mol Biol Rep
123
and 4, while the corresponding results for men are shown in
the text where relevant.
The cumulative occurrence of clinical manifestations
was recorded adhering to the definitions of the ACR clas-
sification criteria used. Typical and prevalent clinical
manifestations were nephritis, malar rash, and arthritis.
Lupus nephritis was defined as persistent proteinuria
[0.5 g per day; this was per se the defining characteristic
for all patients with lupus nephritis, although cellular uri-
nary casts was also an option for defining lupus nephritis.
Malar rash was defined as fixed erythema, flat or raised,
over the malar eminences. Arthritis was defined as non-
erosive arthritis involving 2 or more peripheral joints
characterized by tenderness, swelling, or effusion. Sero-
logically, the presence at some time during the course of
disease of anti-dsDNA antibodies (Abs), anti-Smith Abs,
and anti-U1-small nuclear ribonucleoprotein (RNP) Abs
was recorded. The determination of anti-dsDNA Abs by
the EliA test (Pharmacia Diagnostics�, Freiburg, Germany)
was performed according to the manufacturer’s instruc-
tions. Abs against the extractable nucleoproteins Smith and
U1-RNP were determined using enzyme-linked immuno-
sorbent assays.
A gender-matched group of controls comprising a total
of 443 individuals (412 women and 31 men, Table 1) were
randomly selected (using proc surveyselect in SAS v9.1.3,
seed = 1234567) from a larger group of previously geno-
typed participants from the Copenhagen Aging and Midlife
Biobank (CAMB) [34]. The CAMB data collection took
place in 2009–2011 and had a particular emphasis on the
earlier stages of the aging process, including biological,
psychological and social variables collected by physical
and cognitive testing, clinical examination, blood samples,
and an extensive questionnaire.
The study was approved by The Committees on Bio-
medical research Ethics for the capital region of Denmark
(KF 01-095/03).
Single nucleotide polymorphisms
Single nucleotide polymorphisms were genotyped using an
already existing, in-house assay [35], based on represen-
tative SNPs randomly selected from the dbSNP database
[36] at the time of assay development. Only biallelic SNPs
with a reported heterozygote frequency of at least 1 % in
persons of European ancestry were included. After exclu-
sion of the rs5743781 polymorphism that failed initial
quality controls, a total of eight SNPs located in the human
TLR7, TLR8, and TLR9 genes were included for analyses
(Table 2). Briefly, allele-specific primers were labelled in
an allele-specific primer extension (ASPE) reaction, using
polymerase chain reaction-amplified SNP-sites as their
target sequences. The labelled ASPE-primers were subse-
quently hybridized to MicroPlex-xTAG beadsets for
detection and counting on the Luminex platform (Luminex
Corporation, Austin, TX, USA). All genotypings were
carried out randomized and blinded to the technician per-
forming the genotyping.
Statistical analyses
Hardy–Weinberg equilibrium (HWE)-analysis of genotype
data was performed using an exact test in PowerMarker
v.3.25 [37]. For all other statistical analyses, exact tests in
SAS v.9.1.3 (SAS Institute Inc., Cary, NC, USA) was
employed. Odds ratios (OR) were calculated with exact
confidence intervals (CI) of 95 %. Association trends were
tested by the Cochran–Armitage test for trend. All tests
were stratified by gender and the level of significance was
set at P B 0.05.
Results
Association between investigated SNPs and occurrence
of SLE
In the investigated cohort, we found TLR7-rs3853839 to be
associated with SLE (Table 2). Carriage of the G-allele of
TLR7-rs3853839 was associated with the occurrence of
SLE in women (G vs. C, P = 0.008, OR 1.60, 95 % CI
0.94–2.21), as well as in men (G vs. C, P = 0.02, OR 4.50,
95 % CI 1.18–16.7) (data not shown). Additionally,
Cochran–Armitage trend analysis indicated a gene dose
effect with respect to the minor allele (G) of rs3853839 and
Table 1 Demographic characteristics of systemic lupus erythema-
tosus-patients and healthy controls
Variable Men Women
Patients (n = 142; 10 men/132 women):
Age at time of inclusion (years), mean
(range)
44 (21–67) 46 (21–76)
Age at diagnosis (years), mean (range) 32 (16–51) 31 (11–69)
Clinical manifestations
Malar rash [n (%)] 2 (20) 75 (57)
Arthritis [n (%)] 7 (70) 88 (67)
Nephritis [n (%)] 5 (50) 63 (48)
Serological factors
Anti-DNA antibodies [n (%)] 7 (70) 112 (85)
Anti-Smith antibodies [n (%)] 1 (10) 12 (9)
Anti-U1RNP [n (%)] 0 17 (13)
Healthy controls (n = 443; 31 men/412 women):
Age at time of analysis (years), mean
(range)
57 (50–61) 56 (50–61)
Mol Biol Rep
123
risk of SLE in women (P = 0.009). Applying the Cochran–
Armitage trend analysis, the rs3764879 polymorphism
located in the 50-region of TLR8 could also be associated
with SLE in women (P = 0.05) (Table 2). Carriage of the
G-allele of TLR8-rs3764879 was associated with the
occurrence of SLE in women (G vs. C, P \ 0.05, OR 1.36,
95 % CI 0.99–1.86), but not with statistical significance in
men (G vs. C, P = 0.06, OR 4.00, 95 % CI 0.90–17.3),
although the effect appeared stronger in men than in
women. None of the other investigated SNPs showed
associations to the occurrence of SLE (Table 2).
None of the investigated SNPs deviated significantly
from HWE in the control group (data not shown).
Association between investigated SNPs and clinical
parameters
Within the SLE cohort, TLR7-rs3853839 was associated
with lupus nephritis in women (Table 3). Homozygous
carriage of the G-allele of TLR7-rs3853839 significantly
increased the occurrence of lupus nephritis in women (G/G
vs. C/C and C/G, P = 0.007, OR 11.3, 95 % CI 1.47–504),
but not in men (data not shown). The rs3853839 poly-
morphism was not associated with malar rash, arthritis, nor
any of the autoantibodies investigated (Tables 3, 4).
The TLR9-rs187084 polymorphism was associated with
malar rash in women (C vs. T, P = 0.04, OR 1.73, 95 % CI
1.00–2.99), and a statistically significant risk trend was also
seen in women (P = 0.04). No associations could be
detected for the remaining SNPs and clinical manifesta-
tions (Table 3).
Association between investigated SNPs and serological
parameters
Autoantibodies against dsDNA were associated with the
rs179008 polymorphism of TLR7, in women (Table 4),
and with the rs5741833 polymorphism of TLR8 in men.
The minor allele of rs179008 was positively associated
with the presence of anti-dsDNA Abs (T vs. A, P = 0.03,
OR 3.73, 95 % CI 1.10–19.6) in women, and a statistically
significant risk trend was observed (P = 0.03). In men, the
minor allele of rs5741833 was negatively associated with
the presence of anti-dsDNA Abs (T vs. C, P = 0.04, OR
0.08, 95 % CI 0.005–1.19) (data not shown).
None of the investigated SNPs showed associations to
the occurrence of anti-Smith Abs in the SLE cohort
(Table 4).
Anti-U1-RNP Abs were associated with the rs187084
and rs5743836 polymorphisms of TLR9, in women
(Table 4). The minor allele of rs187084 was negatively
associated with the presence of anti-U1-RNP Abs (C vs. T,
P = 0.01, OR 0.31, 95 % CI 0.10–0.81), and a statisticallyTa
ble
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mo
ter
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26
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48
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ter
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.24
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.05
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74
40
88
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TR
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.96
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(0.5
1–
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.39
–1
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)0
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R9
rs5
74
38
36
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mo
ter
TC
30
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01
50
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25
20
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Mol Biol Rep
123
Ta
ble
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)
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Mo
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ue
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00
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–2
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.00
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(0.1
4–
8.5
3)
0.6
5
TL
R7
rs3
85
38
39
CG
43
25
10
.19
63
61
89
0.2
86
0.1
1,
1.6
4(0
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–3
.04
)0
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,1
.24
(0.5
8–
2.6
4)
0.0
07
,1
1.3
3(1
.47
–5
04
)0
.13
TL
R8
rs5
74
18
83
CT
41
25
30
.22
54
21
83
0.1
90
0.5
5,
0.8
1(0
.43
–1
.54
)0
.47
,0
.73
(0.3
4–
1.5
8)
1.0
0,
1.1
0(0
.14
–8
.53
)0
.55
TL
R8
rs3
76
48
79
CG
35
30
40
.27
52
52
99
0.3
73
0.1
1,
1.5
7(0
.90
–2
.72
)0
.22
,1
.56
(0.7
4–
3.3
1)
0.1
4,
2.7
1(0
.70
–1
2.6
2)
0.1
1
TL
R8
rs5
74
40
88
GC
50
17
20
.15
24
61
70
0.1
35
0.7
4,
0.8
7(0
.41
–1
.83
)1
.00
,0
.97
(0.4
2–
2.2
4)
–0
.72
TL
R9
rs1
87
08
4T
C2
92
81
20
.37
72
13
66
0.3
81
1.0
0,
1.0
2(0
.60
–1
.73
)0
.37
,1
.45
(0.6
7–
3.1
4)
0.2
1,
0.5
0(0
.14
–1
.57
)1
.00
TL
R9
rs5
74
38
36
TC
54
13
20
.12
35
11
20
0.0
95
0.5
6,
0.7
5(0
.31
–1
.75
)0
.83
,0
.85
(0.3
3–
2.1
5)
–0
.56
Mal
arra
sh
TL
R7
rs2
30
22
67
TG
52
50
0.0
44
67
80
0.0
53
0.7
8,
1.2
3(0
.34
–4
.91
)0
.78
,1
.24
(0.3
3–
5.1
1)
–0
.78
TL
R7
rs1
79
00
8A
T3
71
91
0.1
84
46
24
50
.22
70
.45
,1
.30
(0.6
8–
2.5
2)
0.7
2,
1.1
7(0
.54
–2
.55
)0
.23
,4
.00
(0.4
3–
19
3)
0.4
8
TL
R7
rs3
85
38
39
CG
33
19
50
.25
44
62
45
0.2
27
0.6
6,
0.8
6(0
.47
–1
.58
)0
.72
,0
.87
(0.4
1–
1.8
6)
0.7
5,
0.7
4(0
.16
–3
.42
)0
.68
TL
R8
rs5
74
18
83
CT
37
18
20
.19
34
62
54
0.2
20
0.6
5,
1.1
8(0
.62
–2
.28
)0
.72
,1
.17
(0.5
4–
2.5
5)
0.7
0,
1.5
5(0
.21
–1
7.7
)0
.65
TL
R8
rs3
76
48
79
CG
26
26
50
.31
63
43
38
0.3
27
0.8
9,
1.0
5(0
.60
–1
.84
)1
.00
,1
.01
(0.4
8–
2.1
4)
0.7
8,
1.2
4(0
.33
–5
.11
)0
.89
TL
R8
rs5
74
40
88
GC
46
92
0.1
14
50
25
00
.16
70
.29
,1
.55
(0.7
2–
3.4
8)
0.0
8,
2.1
0(0
.87
–5
.24
)–
0.2
8
TL
R9
rs1
87
08
4T
C2
72
55
0.3
07
23
39
13
0.4
33
0.0
4,
1.7
3(1
.00
–2
.99
)0
.07
,2
.03
(0.9
4–
4.4
2)
0.2
0,
2.1
8(0
.67
–8
.29
)0
.04
TL
R9
rs5
74
38
36
TC
44
13
00
.11
46
11
22
0.1
07
0.8
5,
0.9
3(0
.40
–2
.20
)0
.66
,0
.78
(0.3
1–
1.9
9)
–1
.00
Art
hri
tis
TL
R7
rs2
30
22
67
TG
42
20
0.0
23
77
11
00
.06
30
.23
,2
.87
(0.6
0–
27
.1)
0.2
2,
3.0
0(0
.61
–2
8.9
)–
0.2
2
TL
R7
rs1
79
00
8A
T2
61
62
0.2
27
57
27
40
.19
90
.63
,0
.84
(0.4
4–
1.6
7)
0.5
7,
0.7
9(0
.35
–1
.78
)1
.00
,1
.00
(0.1
4–
11
.5)
0.6
3
TL
R7
rs3
85
38
39
CG
29
12
30
.20
55
03
17
0.2
56
0.4
4,
1.3
4(0
.70
–2
.64
)0
.35
,1
.47
(0.6
5–
3.3
8)
1.0
0,
1.1
8(0
.25
–7
.43
)0
.47
TL
R8
rs5
74
18
83
CT
26
17
10
.21
65
72
65
0.2
05
0.8
7,
0.9
3(0
.48
–1
.86
)0
.57
,0
.79
(0.3
5–
1.7
8)
0.6
6,
2.5
9(0
.28
–1
25
.3)
0.8
7
TL
R8
rs3
76
48
79
CG
22
19
30
.28
43
84
01
00
.34
10
.40
,1
.30
(0.7
2–
2.3
9)
0.4
7,
1.3
2(0
.60
–2
.90
)0
.54
,1
.75
(0.4
2–
10
.4)
0.6
6
TL
R8
rs5
74
40
88
GC
32
12
00
.13
66
42
22
0.1
48
0.8
5,
1.1
0(0
.50
–2
.53
)1
.00
,1
.00
(0.4
2–
2.4
9)
–0
.85
TL
R9
rs1
87
08
4T
C1
52
18
0.4
20
35
43
10
0.3
58
0.3
5,
0.7
7(0
.44
–1
.35
)0
.57
,0
.78
(0.3
4–
1.7
7)
0.2
9,
0.5
8(0
.19
–1
.84
)0
.34
TL
R9
rs5
74
38
36
TC
36
80
0.0
91
69
17
20
.11
90
.54
,1
.35
(0.5
5–
3.7
0)
0.8
2,
1.2
4(0
.46
-3.6
0)
–0
.55
Gen
e,P
RR
-gen
eto
wh
ich
the
SN
Pb
elo
ng
s;rs
#,
Ref
SN
PS
NP
-id
enti
fica
tio
nco
de
asap
pli
edin
the
pu
bli
cn
ucl
eic
acid
po
lym
orp
his
md
atab
ases
atN
CB
I;‘‘
p’’
des
ign
ates
the
maj
or
alle
lean
d
‘‘q
’’th
em
ino
ral
lele
(in
Eu
rop
ean
po
pu
lati
on
s).M
AF
min
or
alle
lefr
equ
ency
,O
Ro
dd
sra
tio
,C
Ico
nfi
den
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terv
al,C
–A
tren
dC
och
ran
–A
rmit
age
tren
dte
st.V
alu
esin
bo
ldsi
gn
ifica
ntl
yd
evia
te
fro
mth
en
ull
-hy
po
thes
is(a
B0
.05
)
Mol Biol Rep
123
Ta
ble
4A
sso
ciat
ion
sb
etw
een
sele
cted
po
lym
orp
his
ms
inT
LR
7,
TL
R8
,an
dT
LR
9an
dan
ti-D
NA
,an
ti-S
mit
h(S
m),
and
anti
-U1
-rib
on
ucl
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rote
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NP
)an
tib
od
ies
infe
mal
eS
LE
pat
ien
ts.
Gen
ers
#A
nti
bo
dy
pre
sen
tn
ever
An
tib
od
yp
rese
nt
ever
All
ele
test
(pv
s.q
)D
om
inan
tm
od
el
(pp
vs.
pq
)
Rec
essi
ve
mo
del
(pp
/pq
vs.
)
C–
Atr
end
pq
pp
pq
MA
Fp
pp
qM
AF
P,
OR
(95
%C
I)P
,O
R(9
5%
CI)
P,
OR
(95
%C
I)P
val
ue
An
ti-d
sDN
A
TL
R7
rs2
30
22
67
TG
16
40
0.1
00
10
39
00
.04
00
.12
,0
.38
(0.1
0–
1.7
7)
0.1
1,
0.3
5(0
.09
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.76
)–
0.1
1
TL
R7
rs1
79
00
8A
T1
73
00
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56
64
06
0.2
32
0.0
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3(1
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(1.0
4–
22
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–0
.03
TL
R7
rs3
85
38
39
CG
11
81
0.2
50
68
35
90
.23
70
.84
,0
.93
(0.4
1–
2.2
8)
0.6
3,
0.7
9(0
.27
–2
.36
)1
.00
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.66
(0.2
1–
76
.6)
1.0
0
TL
R8
rs5
74
18
83
CT
10
82
0.3
00
73
35
40
.19
20
.14
,0
.55
(0.2
5–
1.3
0)
0.2
2,
0.5
3(0
.18
–1
.57
)0
.22
,0
.33
(0.0
4–
3.9
8)
0.1
4
TL
R8
rs3
76
48
79
CG
11
63
0.3
00
49
53
10
0.3
26
0.8
6,
1.1
3(0
.52
–2
.58
)0
.47
,1
.57
(0.5
4–
4.6
5)
0.4
2,
0.5
6(0
.13
–3
.48
)0
.85
TL
R8
rs5
74
40
88
GC
12
80
0.4
00
84
26
20
.13
40
.33
,0
.62
(0.2
5–
1.7
1)
0.1
8,
0.5
0(0
.17
–1
.57
)–
0.3
2
TL
R9
rs1
87
08
4T
C7
10
30
.40
04
35
41
50
.37
50
.86
,0
.90
(0.4
3–
1.9
3)
1.0
0,
0.8
6(0
.27
–2
.56
)0
.74
,0
.88
(0.2
1–
5.2
2)
0.8
6
TL
R9
rs5
74
38
36
TC
16
31
0.1
25
89
22
10
.10
70
.78
,0
.84
(0.2
9–
3.0
1)
1.0
0,
1.0
3(0
.29
–4
.66
)0
.28
,0
.17
(0.0
2–
14
.1)
0.7
9
An
ti-S
mit
h
TL
R7
rs2
30
22
67
TG
10
81
20
0.0
50
11
10
0.0
42
1.0
0,
0.8
3(0
.02
–6
.08
)1
.00
,0
.82
(0.0
2–
6.6
4)
–1
.00
TL
R7
rs1
79
00
8A
T7
53
96
0.2
13
84
00
.16
70
.79
,0
.74
(0.1
8–
2.3
5)
1.0
0,
0.8
3(0
.17
–3
.33
)1
.00
,0
.00
(0.0
0–
6.8
)0
.80
TL
R7
rs3
85
38
39
CG
73
39
80
.22
96
42
0.3
33
0.3
1,
1.6
8(0
.59
–4
.43
)0
.54
,1
.55
(0.3
9–
6.1
7)
0.2
3,
2.8
0(0
.25
–1
6.7
)0
.34
TL
R8
rs5
74
18
83
CT
76
39
50
.20
47
41
0.2
50
0.6
0,
1.3
0(0
.40
–3
.65
)0
.76
,1
.23
(0.2
9–
4.8
2)
0.4
4,
2.0
9(0
.04
–2
1.2
)0
.80
TL
R8
rs3
76
48
79
CG
55
55
10
0.3
13
54
30
.41
70
.36
,1
.57
(0.5
9–
4.0
0)
1.0
0,
1.1
8(0
.30
–5
.01
)0
.10
,3
.67
(0.5
4–
18
.0)
0.3
6
TL
R8
rs5
74
40
88
GC
89
29
20
.13
87
50
0.2
08
0.3
6,
1.6
5(0
.45
–4
.99
)0
.31
,2
.05
(0.4
7–
8.1
0)
1.0
0,
0.0
0(0
.00
–3
5.7
)0
.35
TL
R9
rs1
87
08
4T
C4
55
81
70
.38
35
61
0.3
33
0.8
3,
0.8
0(0
.29
–2
.09
)0
.76
,0
.84
(0.2
2–
3.5
7)
1.0
0,
0.5
5(0
.01
–4
.28
)0
.66
TL
R9
rs5
74
38
36
TC
96
22
20
.10
89
30
0.1
25
0.7
4,
1.1
8(0
.21
–4
.36
)0
.71
,1
.33
(0.2
2–
5.8
9)
1.0
0,
0.0
0(0
.00
–3
5.7
)1
.00
An
ti-U
1-R
NP
TL
R7
rs2
30
22
67
TG
10
21
20
0.0
53
16
10
0.0
29
1.0
0,
0.5
2(0
.01
–3
.75
)1
.00
,0
.53
(0.0
1–
4.1
0)
–0
.70
TL
R7
rs1
79
00
8A
T7
33
74
0.1
97
96
20
.29
40
.26
,1
.69
(0.6
7–
3.9
9)
0.4
3,
1.5
8(0
.49
–5
.01
)0
.17
,3
.67
(0.3
0–
27
.8)
0.2
6
TL
R7
rs3
85
38
39
CG
69
38
70
.22
81
04
30
.29
40
.39
,1
.41
(0.5
6–
3.3
0)
1.0
0,
1.0
7(0
.32
–3
.39
)0
.12
,3
.28
(0.4
8–
16
.4)
0.5
4
TL
R8
rs5
74
18
83
CT
75
34
50
.19
38
81
0.2
94
0.1
8,
1.7
4(0
.69
–4
.11
)0
.18
,2
.16
(0.6
8–
6.9
7)
0.5
7,
1.3
6(0
.03
–1
3.3
)0
.26
TL
R8
rs3
76
48
79
CG
52
52
10
0.3
16
86
30
.35
30
.70
,1
.18
(0.5
0–
2.6
6)
1.0
0,
0.9
4(0
.30
–3
.03
)0
.38
,2
.23
(0.3
5–
10
.1)
0.6
9
TL
R8
rs5
74
40
88
GC
86
26
20
.13
29
80
0.2
35
0.1
2,
2.0
3(0
.72
–5
.16
)0
.08
,2
.73
(0.8
2–
8.7
8)
1.0
0,
0.0
0(0
.00
–2
3.7
)0
.11
TL
R9
rs1
87
08
4T
C3
95
71
80
.40
81
16
00
.17
60
.01
,0
.31
(0.1
0–
0.8
1)
0.0
3,
0.2
8(0
.08
–0
.92
)0
.13
,0
.00
(0.0
0–
1.1
3)
0.0
1
TL
R9
rs5
74
38
36
TC
94
18
20
.09
61
07
00
.20
60
.08
,2
.43
(0.8
0–
6.5
9)
0.0
5,
3.2
9(0
.93
–1
0.9
)1
.00
,0
.00
(0.0
0–
23
.7)
0.0
8
Gen
e,g
ene
tow
hic
hth
eS
NP
bel
on
gs;
rs#
,R
efS
NP
SN
P-i
den
tifi
cati
on
cod
eas
app
lied
inth
ep
ub
lic
nu
clei
cac
idp
oly
mo
rph
ism
dat
abas
esat
NC
BI;
‘‘p
’’d
esig
nat
esth
em
ajo
ral
lele
and
‘‘q
’’th
e
min
or
alle
le(i
nE
uro
pea
np
op
ula
tio
ns)
.M
AF
min
or
alle
lefr
equ
ency
,O
Ro
dd
sra
tio
,C
Ico
nfi
den
cein
terv
al,
C–
Atr
end
Co
chra
n–
Arm
itag
etr
end
test
.V
alu
esin
bo
ldsi
gn
ifica
ntl
yd
evia
tefr
om
the
nu
ll-h
yp
oth
esis
(aB
0.0
5)
Mol Biol Rep
123
significant risk trend was also seen (P = 0.01). Conversely,
homozygous carriage of the C-allele of TLR9-rs5743836
significantly increased the occurrence of anti-U1-RNP Abs
in women (C/C vs. T/T and T/C, P \ 0.05, OR 3.29, 95 %
CI 0.93–10.9). No patient with a history of this autoanti-
body was homozygous for the TLR9-rs187084 or TLR9-
rs5743836 polymorphisms.
Discussion
In the present study, we investigated the relationships
between eight SNPs in the human TLR7-, TLR8-, and
TLR9-encoding genes and the occurrence of SLE, lupus
nephritis, malar rash, arthritis, anti-dsDNA-, anti-Smith,
and anti-U1-RNP Abs in a Danish cohort of SLE patients.
Recently, the TLR7-rs3853839 polymorphism was
reported to be associated with increased risk of SLE in
Eastern Asians [15], European Americans, African Amer-
icans, and Amerindian/Hispanics [38].
Interestingly, in this Danish cohort the SLE risk-allele
(G) is the minor allele of the rs3853839 polymorphism,
while this appears to be the major allele in all published
studies pertaining to Asian cohorts. Still, the European
minor allele and the major allele in Asia confer roughly the
same risk of SLE. These observations may contribute to the
understanding of the variations in incidence of SLE
between different populations [39].
A previous study of an Asian population has shown a
particularly high risk for development of SLE in male
carriers of the G-allele of rs3853839, as compared to
female carriers [15]. In our study, a similar pattern was
observed, but the association did not reach statistical sig-
nificance due to low statistical power in the male group
(n = 10) of SLE patients (Table 2). Our study also sug-
gested an association between the TLR7-rs3853839 poly-
morphism and lupus nephritis, a relationship that has not
previously been reported.
The fact that the TLR7-rs3853839 polymorphism
showed significant associations to both SLE occurrence
and nephritis lends further support to the significance of
this polymorphism in SLE pathogenesis.
Except for one study [17], no associations between
SNPs in TLR8 and SLE or SLE-related clinical or para-
clinical parameters have previously been reported. In our
study, the rs3764879 SNP of TLR8 was significantly
associated with SLE occurrence and this particular SNP
has not previously been associated with SLE. The
rs3764879 SNP is located at position -129 relative to the
translation start site in the 50-end region of TLR8 and may
thus be of regulatory importance. In addition to its well-
known role as a sensor of ssRNA, TLR8 has been impli-
cated in bacterial sensing and receptor cross-talk [40], and
the rs3764879 SNP has been associated with development
of pulmonary tuberculosis [41]. Moreover, rs3764879 is in
strong linkage disequilibrium with rs3764880 (Met1Val)
which has also been associated with pulmonary tubercu-
losis [41], and additionally with sensing of Helicobacter
pylori [42], Borrelia burgdorferi [40], and restriction of
HIV disease [43], emphasizing the functional role of
genetic variation in this area of TLR8. However, in our
study, the rs3764879 SNP was not associated with any
other of the investigated parameters, nor was the observed
association strong enough to survive correction for multi-
ple testing. Further analyses are therefore needed to con-
firm this finding.
The rs179008 and rs5741883 polymorphisms of TLR7
and TLR8, respectively, showed associations to anti-
dsDNA Abs in this study. Since TLR7 and TLR8 are not at
present known to be directly involved in the response to
dsDNA, these associations are most likely spurious false-
positive observations.
The rs187084 SNP of TLR9 was negatively associated
with anti-U1-RNP Abs and positively with malar rash,
whereas the rs5743836 SNP of TLR9 was positively
associated with anti-U1-RNP Abs. However, these associ-
ations were not strong and risk of false-positive spurious
findings should be considered.
It is a strength of this study that the controls were rep-
resentative of the general population in Denmark, enabled
by the randomly selected inclusion of gender-matched
samples from the CAMB-cohort [34]. However, due to the
limited size of our study, and the previously presented
evidence of association, we did not apply correction for
multiple testing to any of the association results and they
should consequently be considered with caution.
In conclusion, our study confirms the association
between the rs3853839 polymorphism of TLR7 and SLE in
a Danish cohort, a finding that was further corroborated by
the observation of a simultaneous association between
rs3853839 and lupus nephritis. Furthermore, a possible
association between SLE and the rs3764879 SNP of TLR8
is indicated.
Acknowledgments We thank Pia Grothe Meinke for technical
assistance. We also thank all patients and healthy control subjects for
their participation. The Danish Biotechnology Program, Novo Nor-
disk Foundation, The Danish Rheumatism Association, and The
Lundbeck Foundation are thanked for financial support. The Copen-
hagen Aging and Midlife Biobank has been supported by a generous
grant from the VELUX FOUNDATION. The authors wish to thank
Prof. Palle Holmstrup for establishing and making possible the project
that enabled genotyping of the control samples from the CAMB
cohort used herein. The authors thank the staff at the Institute of
Public Health and the National Research Center for the Working
Environment who undertook the data collection. Further thanks to
Helle Bruunsgaard, Nils-Erik Fiehn, Ase Marie Hansen, Poul Holm-
Pedersen, Rikke Lund, Erik Lykke Mortensen and Merete Osler who
initiated and established the Copenhagen Aging and Midlife Biobank
Mol Biol Rep
123
from 2009 to 2011 together with Kirsten Avlund. The authors
acknowledge the crucial role of the initiators and steering groups of
the Metropolit Cohort, The Copenhagen Perinatal Cohort and The
Danish Longitudinal Study on Work Unemployment and Health.
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