Association of CD209 and CD209L polymorphismswith tuberculosis infection in a Northeastern Brazilian population

Ronaldo Celerino da Silva • Ludovica Segat • Heidi Lacerda Alves da Cruz •

Haiana Charifker Schindler • Lilian Maria Lapa Montenegro • Sergio Crovella •

Rafael Lima Guimaraes

Received: 8 March 2013 / Accepted: 15 May 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Tuberculosis (TB) caused by Mycobacterium

tuberculosis, is major cause of morbidity and mortality

worldwide. So far, many candidate genes have been

investigated for their possible association with TB. Den-

dritic cell-specific intercellular adhesion molecule 3

(ICAM-3) grabbing non-integrin (DC-SIGN) and Liver/

lymph node-specific intercellular adhesion molecule-grab-

bing non-integrin (L-SIGN), encoded by CD209 and

CD209L genes respectively, are known for binding to M.

tuberculosis on human dendritic cells and macrophages. We

screened 4 single nucleotide polymorphisms (SNPs) in the

promoter region of CD209, namely -939G[A (rs735240),

-871A[G (rs735239), -336A[G (rs4804803) and

-139G[A (rs2287886) and tandem repeat polymorphisms

in exon 4 of CD209 and CD209L genes looking for asso-

ciation with TB in a Northeastern Brazilian population (295

subjects, 131 TB patients and 164 healthy controls). The

-139G[A and -939G[A SNPs were associated with

susceptibility to TB, and in particular with pulmonary and

extra-pulmonary forms respectively. The -871A[G and

-336A[G SNPs were associated, the first with protection

to both pulmonary and extra-pulmonary TB, the latter only

with the pulmonary form. An association between GGAG

haplotype and protection to TB infection was also found.

Also tandem repeat polymorphism in CD209L exon 4 was

associated with TB infection. This study provides evidence

of an association between CD209 and CD209L polymor-

phisms and TB development in a Brazilian population,

suggesting that variations in these genes may influence the

protection and susceptibility to infection caused by M.

tuberculosis.

Keywords Polymorphisms � CD209 � CD209L �Tuberculosis

Introduction

Tuberculosis (TB) is a chronic disease, mainly affecting the

lungs, caused by Mycobacterium tuberculosis, which is the

major cause of morbidity and mortality for infectious dis-

eases worldwide [1, 2].

Mycobacterium tuberculosis has the ability to bind to

several cellular receptors, such as dendritic cell-specific

ICAM-grabbing non-integrin (DC-SIGN) and liver/lymph

node-specific ICAM-grabbing non-integrin (L-SIGN)

receptors, which interact with the lipoarabinomannan

(ManLAN) present in the bacillus cell wall, mediating the

recognition, the inflammatory response regency and the

bacillus persistence [3, 4].

CD209 and CD209L genes (both mapping at chromo-

some 19p13.2-3) encode DC-SIGN and L-SIGN respec-

tively. The first one is expressed in macrophages and

dendritic cells (DCs) (DC-SIGN) [5–7], and the latter in

R. C. da Silva (&) � H. L. A. da Cruz � S. Crovella �R. L. Guimaraes

Department of Genetics, Federal University of Pernambuco

(UFPE), Av. Prof. Moraes Rego, 1235 - Cidade Universitaria,

Recife, PE, Brazil

e-mail: ronaldocelerino@yahoo.com.br

R. C. da Silva � H. L. A. da Cruz � S. Crovella � R. L. Guimaraes

Laboratory of Immunopathology Keizo Asami (LIKA), Federal

University of Pernambuco (UFPE), Recife, PE, Brazil

L. Segat

Institute for Maternal and Child Health, IRCCS Burlo Garofolo,

Trieste, Italy

H. C. Schindler � L. M. L. Montenegro

Departament of Immunology, Aggeu Magalhaes Research

Center (CPqAM– FIOCRUZ – Pernambuco), Recife, PE, Brazil

123

Mol Biol Rep

DOI 10.1007/s11033-014-3416-y

endothelial cells (L-SIGN) in various tissues, including the

lungs [4, 6, 8]. These genes share a similar intron–exon

distribution and are organized into three domains: the

N-terminal cytoplasmic tail, the transmembrane domain

and the C-terminal domain; the latter is formed by a

repeated region, containing seven tandem repeats of 23

amino acids and a carbohydrate recognition domain,

involved in recognizing and binding to pathogens [4].

Both DC-SIGN and L-SIGN receptors have been

reported to be involved in the activation of immune

response and immunologic escape of pathogens such as M.

tuberculosis [9]. Additional studies have suggested that

polymorphisms in CD209 and CD209L genes are possibly

involved in the protection and susceptibility to infections

by M. tuberculosis, in different populations [10, 11].

This study aims to evaluate the possible role of poly-

morphisms in CD209 and CD209L genes on the suscepti-

bility to M. tuberculosis infection in a Northeast Brazilian

population.

Materials and methods

Study population

The study population (Table 1) consisted of 295 individ-

uals from the metropolitan area of Recife (Pernambuco

State, Northeast of Brazil), and was divided in two groups:

Healthy controls (HC) 164 healthy individuals of both

sexes, HIV-1 negative, without previous history of tuber-

culosis (all negative for tuberculin test), not related with

patients.

TB patients (TB) 131 individuals of both sexes, with

clinical and/or radiologic evidence of TB, contact history

with carriers, laboratorial confirmation of diagnosis

through baciloscopy or culture, and positive tuberculin test.

Tuberculosis cases were subdivided, according to WHO

classification [12], in pulmonary (pTB) and extra-pul-

monary TB (eTB).

Since it was quite impossible to enroll a sort of M.

tubercolosis exposed uninfected controls, we tried to

choose the best controls in terms of ethnicity and

possibility of similar exposition as TB patients, by enroll-

ing healthy individuals from the same areas (and with same

ethnical background) where patients have been collected.

The Brazilian Northeastern population enrolled for this

study was reported to be derived from an admixture of

African, Caucasian and Native American populations,

estimated at 44, 34 and 22 %, respectively [13].

All subjects, or their parents in case of minor age, gave

the informed consent, under supervision of CPqAM/FIO-

CRUZ Ethics Committee (CEP Registration—55/05).

DNA extraction and genotyping

Genomic DNA extraction was performed from human

peripheral blood samples anti-coagulated with EDTA,

using the commercial kit ‘‘Blood Genomic Prep DNA

Isolation Kit�’’ (Promega, Madison MD), following the

manufacture’s instructions.

Four SNPs at the CD209 promoter were analyzed:

-139G[A (rs2287886), -336A[G (rs4804803),

-871A[G (rs735239) and -939G[A (rs735240). The

genotypes were assessed by allele-specific probes (Taq-

Man�, Applied Biosystems), using the real time PCR

platform ABI� 7500 (Applied Biosystems). These SNPs

were selected according to their potential functional effect

or previous associations reported [11, 14, 15]. Amplifica-

tions were accomplished with the following thermal con-

ditions: initial denaturation at 95 �C for 10 min, followed

by 40 cycles at 92 �C for 30 s and 60 �C for 1 min. Some

samples were randomly chosen and sequenced in order to

double-check the obtained results.

CD209 and CD209L exon 4 tandem-repeat polymor-

phisms were analyzed by conventional PCR and gel elec-

trophoresis using methods previously described [16, 17].

Two independent operators performed in blind the geno-

typing, sequencing of randomly chosen samples was also

performed.

Unfortunately, due to the poor quality of some of the

biological samples available for DNA extraction, not all

samples amplified for all tested polymorphisms, and the

number of subjects genotyped was not 100 % for each

SNP.

Table 1 Population

demographical characteristicsCharacteristics Healthy controls TB patients

Total pTB eTB

n (total = 295) 164 131 95 36

Mean age: years ± SD 21.64 ± 2.42 25.97 ± 18.27 24.75 ± 17.56 28.78 ± 19.45

Sex male/female: n (%): 57/107 (34.8/65.2) 76/55 (58.0/42.0) 60/35 (63.2/36.8) 16/20 (44.4/55.6)

Mol Biol Rep

123

Statistical analysis

Alleles and genotypes frequencies were calculated by

direct counting and the haplotypes were obtained using the

Arlequin software 3.11 [18]. Hardy–Weinberg equilibrium

(EHW) was evaluated by v2 test. Fisher exact test was used

to evaluate the eventual genetic association. Odds ratio

(OR) and 95 % confidence intervals (95 % CI) have also

been calculated. When calculating OR, the alleles and

corresponding homozygous genotypes, as well as the

haplotype with major frequency in the control group have

been selected as reference (OR = 1.00), and the other ORs

have been calculated relative to that reference (Fisher’s

exact test, 2 9 2 contingency tables, degrees of free-

dom = 1). Statistical analyses were performed using the R

software 2.11.1 [19] and the p values were adjusted by sex

with SNPStats [20]. Bonferroni’s correction for multiple

comparisons was applied considering two independent

hypotheses, namely: the association between TB and

CD209 polymorphisms and the association between TB

and CD209L polymorphisms; the threshold for statistical

significance was thus set at 0.025.

Results

CD209 polymorphisms

The allele and genotype frequencies of the four SNPs

analyzed at the CD209 promoter region were in HW

equilibrium, both for patients and HCs, except those for

SNP -871A[G and -336A[G in total and pulmonary TB,

and -939G[A in HCs.

Significant differences in allelic and genotypic distri-

butions among the analyzed groups were observed for all

SNPs (Table 2).

For -939G[A SNP, we observed that the -939A allele

was significantly more frequent in eTB than in HCs (54.8

vs. 36.6 %, p = 0.01, OR 2.10, 95 % CI 1.17–3.79), sug-

gesting an association with susceptibility to develop eTB;

the -939G allele was significantly associated with pro-

tection against eTB accordingly to a recessive genetic

model (GG vs. GA?AA, p = 0.017, OR 0.35, 95 % CI

0.12–0.89).

When the -871A[G SNP was analyzed, we observed

that the -871G allele was significantly more frequent in

HCs (41.5 %) than in TB in general (24.5 %,

p = 9.354e-05), pulmonary TB (25.6 %, p = 0.0008) and

eTB (20.5 %, p = 0.008), indicating a protective effect

toward TB development (OR = 0.46, OR = 0.49 and

OR = 0.36, respectively, see Table 2). The -871GG

genotype was significantly more frequent in HCs (16.4 %)

than in TB in general (10.8 %, p = 0.013) and the

-871AG heterozygous genotype was significantly more

frequent in HCs (50.3 %) than in TB in general (27.5 %,

p = 1.987e-05) and pulmonary TB (26.3 %,

p = 5.606e-05). These results indicate that the presence of

-871G allele, both in heterozygosis and homozygosis, may

exert a protective effect against TB (OR = 0.30 and

OR = 0.36) while the A allele confers increased suscep-

tibility toward all types of TB when in homozygosis (AA

vs. GA?AA, p \ 0.01).

For the -336A[G SNP, the G allele was more frequent

in HCs (23.7 %) than in TB in general (15.9 %), in pul-

monary TB (15.9 %) and in eTB (16.1 %), but these dif-

ferences were not statistically significant (p [ 0.025). The

A allele was associated with increased disease suscepti-

bility for pulmonary TB when in homozygosis (AA vs.

GG?AG, p = 0.007, OR = 2.06, 95 % CI 1.18–3.64).

For the -139G[A SNP, the A allele was significantly

more frequent in pulmonary TB (40.8 %, p = 0.017) than

in HCs (30.2 %), indicating increased susceptibility toward

pulmonary TB (OR = 1.59) while the G allele was asso-

ciated with protection toward pulmonary TB, when in

homozygosis (GG vs. GA?AA, p = 0.016, OR = 0.50,

95 % CI 0.28–0.89).

The four SNPs at the CD209 promoter (-939G[A,

-871A[G, -336A[G and -139G[A) were not in linkage

disequilibrium, nor in patients nor in controls (D0\ 0.85)

and combined into 16 haplotypes (Table 3). The GGAG

and GGGG haplotypes were more frequent in HCs than in

TB (9.2 vs. 2.2 and 4.6 vs. 0 %, p = 0.003 and p = 0.002,

respectively), indicating a possible protective effect

towards TB development. A similar trend (although not

statistically significant, p = 0.029) was seen for the GGAA

haplotype, while the AAAA haplotype was more frequent

in TB patients than controls (10.8 vs 3.9 %) but also in this

case the difference did not reach the statistical significance

(p = 0.028).

The distribution of CD209 exon 4 tandem repeat poly-

morphism was in HW equilibrium in both groups. We

found low genetic variability in this region: two alleles (6

and 7 repetitions) and two genotypes (7/6 and 7/7) were

observed, but no significant differences were evidenced

between patients and controls, although the 6 repetition

allele was only found in controls, and in none of the

patients (p = 0.027) (Table 4).

CD209L polymorphism

The CD209L exon 4 showed a considerable variability in

TB patients and controls: we observed seven different

alleles, ranging from 4 to 10 repeats, whose frequencies

were all in HW equilibrium in both patients and controls

(Table 5). The allele with 7 repeats and the homozygous

7/7 genotype, being more frequent among controls, were

Mol Biol Rep

123

Table 2 Allele and genotype frequencies of CD209 promoter SNPs in TB patients and healthy controls from Northeastern Brazil

SNPs TB patients HC n (%) p value, OR (95 % CI)

Total n

(%)

pTB n

(%)

eTB n

(%)

Total vs HC pTB vs HC eTB vs HC

-939G[A

G 137 (57.1) 109

(61.2)

28 (45.2) 199

(63.4)

Reference Reference Reference

A 103 (42.9) 69 (38.8) 34 (54.8) 115

(36.6)

0.137, 1.30 (0.91–1.86) 0.698, 1.09 (0.73–1.63) 0.010*, 2.10

(1.17–3.79)

GG 40 (33.3) 33 (37.1) 7 (22.6) 72 (45.9) Reference Reference Reference

GA 57 (47.5) 43 (48.3) 14 (45.2) 55 (35.0) 0.031, 1.86 (1.05–3.30) 0.082, 1.70 (0.92–3.15) 0.059, 2.60

(0.91–8.16)

AA 23 (19.2) 13 (14.6) 10 (32.3) 30 (19.1) 0.392, 1.38 (0.67–2.83) 1.000, 0.94 (0.40–2.16) 0.026, 3.39

(1.05–11.57)

GA/

AA

80 (66.7) 56 (62.9) 24 (77.4) 85 (54.1) 0.037, 1.69 (1.01–2.86) 0.227, 1.43 (0.82–2.54) 0.017*, 2.89

(1.12–8.42)

-871A[G

A 154 (75.5) 119

(74.4)

35 (79.5) 179

(58.5)

Reference Reference Reference

G 50 (24.5) 41 (25.6) 9 (20.5) 127

(41.5)

9.354e-05*, 0.46

(0.30–0.69)

0.0008*, 0.49 (0.31–0.75) 0.008*, 0.36

(0.15–0.80)

AA 63 (61.8) 49 (61.3) 14 (63.6) 51 (33.3) Reference Reference Reference

GA 28 (27.5) 21 (26.3) 7 (31.8) 77 (50.3) 1.987e-05*, 0.30

(0.16–0.54)

5.606e-05*, 0.29

(0.14–0.55)

0.031, 0.33

(0.11–0.96)

GG 11 (10.8) 10 (12.5) 1 (4.5) 25 (16.4) 0.013*, 0.36 (0.14–0.84) 0.047, 0.42 (0.16–1.02) 0.058, 0.15

(0.003–1.08)

GA/

GG

39 (38.2) 31 (38.8) 8 (36.4) 102

(66.7)

1.085e-05*, 0.31

(0.18–0.54)

5.195e-05*, 0.32

(0.17–0.57)

0.009*, 0.29

(0.10–0.79)

-336A[G

A 190 (84.1) 138

(84.1)

52 (83.9) 232

(76.3)

Reference Reference Reference

G 36 (15.9) 26 (15.9) 10 (16.1) 72 (23.7) 0.029, 0.61 (0.38–0.97) 0.056, 0.61 (0.35–1.02) 0.242, 0.62

(0.27–1.31)

AA 83 (73.5) 62 (75.6) 21 (67.7) 87 (57.2) Reference Reference Reference

GA 24 (21.2) 14 (17.1) 10 (32.3) 58 (38.2) 0.004*, 0.43 (0.23–0.79) 0.001*, 0.34 (0.16–0.68) 0.543, 0.71

(0.28–1.73)

GG 6 (5.3) 6 (7.3) 0 (0.0) 7 (4.6) 1.000, 0.90 (0.24–3.27) 0.776, 1.20 (0.32–4.40) 0.347, 0.00

(0.00–3.12)

GA/

GG

30 (26.5) 20 (24.4) 10 (32.3) 65 (42.8) 0.007*, 0.48 (0.27–0.84) 0.007*, 0.43 (0.22–0.81) 0.321, 0.64

(0.25–1.53)

-139G[A

G 151 (62.9) 103

(59.2)

48 (72.7) 229

(69.8)

Reference Reference Reference

A 89 (37.1) 71 (40.8) 18 (27.3) 99 (30.2) 0.087, 1.36 (0.94–1.97) 0.017*, 1.59 (1.06–2.38) 0.768, 0.87

(0.45–1.61)

GG 45 (37.5) 29 (33.3) 16 (48.5) 82 (50.0) Reference Reference Reference

GA 61 (50.8) 45 (51.7) 16 (48.5) 65 (39.6) 0.042, 1.71 (1.00–2.92) 0.023*, 1.95 (1.07–3.61) 0.563, 1.26

(0.54–2.92)

AA 14 (11.7) 13 (14.9) 1 (3.0) 17 (10.4) 0.408, 1.50 (0.62–3.57) 0.076, 2.15 (0.85–5.38) 0.466, 0.30

(0.01–2.23)

GA/

AA

75 (62.5) 58 (66.7) 17 (51.5) 82 (50.0) 0.040, 1.66 (1.00–2.77) 0.016*, 1.99 (1.13–3.58) 1.000, 1.06

(0.47–2.42)

* Statistically significant p value

OR odds ratio, 95 % CI confidence intervals, nc not calculable, TB pulmonary tuberculosis, eTB extra-pulmonary tuberculosis, HC healthy

controls

Mol Biol Rep

123

adopted as reference (OR = 1.00) and compared with all

other alleles and genotypes.

The allele with 5 repeats was significantly more frequent

in HCs (13.1 %) than in TB in general (6.5 %, p = 0.025)

and in pulmonary TB (4.4 %; p = 0.006), and associated

with protection to TB (OR = 0.46 for TB in general, and

OR = 0.30 for pTB). Although not statistically significant,

similar trends of association were also seen for the alleles

with 4 and 6 repetitions.

Conversely, the allele with 9 repeats was significantly

more frequent in TB in general (21.7 %, p = 0.002), in

pulmonary TB (20.9 %, p = 0.011) and also in extra-pul-

monary TB (23.6 %, p = 0.006) than in HCs (7.9 %),

indicating association with susceptibility to TB infection

(OR = 2.52 for TB in general, OR = 2.31 for pTB and

OR = 3.08 for eTB).

When analyzing the genotypes, only one significant

association was observed: the 6/5 genotype was signifi-

cantly more frequent in HCs (7.0 %) than in TB in general

(0.9 %; p = 0.014) and in pulmonary TB (0.0 %,

p = 0.007), being associated with protection to TB

(OR = 0.09 for TB in general). Also the 6/6 genotype was

more frequent in HCs than in TB in general and in pTB, but

the difference was not statistically significant considering

Bonferroni’s correction.

We also performed an analysis comparing all the

homozygous genotypes grouped together, with all the

heterozygous grouped together as well, but no significant

differences were observed (p [ 0.025).

Discussion

CD209 promoter region has different binding sites for

transcription factors such as AP-1, Sp-1, Ets-1 and NF-kb

[21]. Polymorphisms in proximity of these sites, can affect

the transcriptional levels, influencing the amount of

expressed protein [21]. So, it was hypothesized that SNPs

in nearby regions such as the -939G[A, -871A[G,

-336A[G and -139G[A SNP, could modulate gene

transcription, increasing and/or decreasing the expression

level of DC-SIGN [22].

In our study, the -139G[A SNP, was significantly

associated with extra-pulmonary TB, the -139G allele in

homozygosis being protective, and the -139A allele con-

ferring increased susceptibility. The -139A allele has been

previously associated with a greater resistance to invasive

pulmonary aspergillosis [23], protection against HTLV-1

infection [24] and predisposition to sever forms of tick-

Table 3 Haplotypes frequencies of CD209 promoter SNPs in TB

patients and healthy controls from Northeastern Brazil

Haplotypes TB patients

n (%)

Healthy

controls

n (%)

p value, OR

(95 % CI)

GAAG 59 (31.7) 84 (29.8) Reference

GGAG 4 (2.2) 26 (9.2) 0.003*, 0.22 (0.05–0.68)

AAAG 11 (5.9) 23 (8.2) 0.436, 0.68 (0.28–1.59)

AGAG 14 (7.5) 21 (7.4) 1.000, 0.95 (0.41–2.14)

GAAA 27 (14.5) 20 (7.1) 0.064, 1.91 (0.94–3.97)

AGAA 18 (9.7) 18 (6.4) 0.353, 1.42 (0.64–3.17)

AGGG 5 (2.7) 16 (5.7) 0.154, 0.45 (0.12–1.37)

GGAA 2 (1.1) 14 (5.0) 0.029, 0.20 (0.02–0.94)

GAGG 11 (5.9) 13 (4.6) 0.824, 1.20 (0.45–3.14)

GGGG 0 (0.0) 13 (4.6) 0.002*, nc (nc–0.49)

AAAA 20 (10.8) 11 (3.9) 0.028, 2.57 (1.08–6.43)

GAGA 3 (1.6) 7 (2.5) 0.741, 0.61 (0.10–2.82)

AAGG 9 (4.8) 5 (1.8) 0.156, 2.55 (0.72–10.19)

GGGA 2 (1.1) 5 (1.8) 0.701, 0.57 (0.05–3.63)

AGGA 1 (0.5) 5 (1.8) 0.402, 0.29 (0.01–2.66)

AAGA 0 (0.0) 1 (0.4) 1.000, nc (nc–56.14)

OR odds ratio, 95 % CI confidence intervals, nc not calculable, TB

tuberculosis patients

* Statistically significant p value

Table 4 Allele and genotype frequencies of CD209 exon 4 tandem repeat polymorphism in TB patients and healthy controls from Northeastern

Brazil

TB patients HC n (%) p value. OR (95 % CI)

Total n (%) pTB n (%) eTB n (%) Total vs. HC pTB vs. HC eTB vs. HC

Alleles

7 208 (100.0) 158 (100.0) 50 (100.0) 332 (97.6) Reference Reference Reference

6 0 (0.0) 0 (0.0) 0 (0.0) 8 (2.4) 0.027, nc (0.00–0.95) 0.060, nc (0.00–1.25) 0.603, nc (0.00–4.020

Genotypes

7/7 104 (100.0) 79 (100.0) 25 (100.0) 166 (97.6) Reference Reference Reference

7/6 0 (0.0) 0 (0.0) 0 (0.0) 4 (2.4) 0.301, nc (0.00–2.47) 0.310, nc (0.00–3.26) 1.000, nc (0.00–10.56)

OR odds ratio, 95 % CI confidence intervals, nc not calculable, TB pulmonary tuberculosis, eTB extra-pulmonary tuberculosis, HC healthy

controls

* Statistically significant p value

Mol Biol Rep

123

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Mol Biol Rep

123

borne encephalitis [25]. On other hand, the -139G allele

was associated with increased risk of development of

human cytomegalovirus (hCMV) reactivation and disease

[26].

The -336A allele, in homozygosis, was associated in

our study with increased susceptibility to the pulmonary

form of the disease. Similar results were achieved by

Vannberg et al. [27], who reported that -336G variant

allele was associated with protection against TB in indi-

viduals from Sub-Saharan Africa. The -336A[G SNP is

located 212 base pairs from the major transcription start

site toward the 50 end of the gene [21], and the -336A

allele has been shown to affect a Sp1-like binding site and

to modulate transcriptional activity in vitro by increasing

the levels of expression [22]. According to our and

Vannberg et al. [27] results, we could hypothesize that

higher levels of DC-SIGN, as in the presence of -336A

allele, could favor TB development, while lower levels

associated with -336G allele, could be protective; this is

consistent with the evidence that M. tuberculosis can uti-

lize DC-SIGN binding to suppress the protective pro-

inflammatory immune response.

However, Barreiro et al. [28], in a study conducted

among TB patients from South Africa, observed that

-336A allele was associated with disease protection and

Selvaraj et al. [11] showed that the 336GG genotype was

associated with susceptibility to HIV–TB co-infection in

patients from South India [11], while other studies in

Colombian [29], Tunisian [17] and Chinese [30] popula-

tions lacked to find any association between the -336A[G

SNP and TB.

For the -871A[G SNP, in our study the presence of

-871 G allele, both in heterozygosis and homozygosis, was

associated with a protective effect against TB, while the A

allele conferred increased susceptibility towards all types of

TB when in homozygosis, in accordance with previous

findings of Barreiro et al. [28]. On the other hand, Zheng

et al. [30], studying a Chinese population with TB, found no

association between the disease and the -871 SNP.

Another association was found for the -939 SNP: the

-939G allele was associated with protection to extrapul-

monary TB. In the literature, only one report describing

relationships between -939SNP and TB infection. The G

allele and GG genotype were associated with risk to TB in

an Indonesian population [31]. Another report concerning

the association of -939G allele with hCMV reactivation

and disease [26] was found.

The role of CD209 promoter region SNPs in the mod-

ulation of receptor expression seems to be contradictory.

However, previous studies have considered only the role

played by a single particular SNP in CD209 expression, not

taking into account that other SNPs can contemporarily and

synergistically modulate gene expression; thus the study of

several SNPs combined in the form of haplotypes or

combined genotypes could provide more reliable infor-

mation. Something similar was done by Mezger et al. [26]:

studying the expression of CD209 on immature DCs from

HCs they found that individuals with both genotypes

-139AA and -939GG had high expression levels of

CD209 than individuals with genotype -139GG and

-939AA [26].

Discrepancies between the studies mentioned above

could in part be explained by ethnicity, since the fre-

quencies of CD209 SNPs vary in different populations: for

example, when considering the -871 SNP (rs735239)

according to the NCBI and HapMap databases [32], the

frequency of the ancestral ‘‘A’’ allele is 0.62 in European

(CEU), 0.84 in Asian (HCB), 0.81, Asian (JPT) and 0.99 in

Sub-Saharan African (YRI). As Barreiro et al. [28]

hypothesize, the geographic distribution of CD209 alleles,

together with their phylogenetic status, could be the result

of host genetic adaptation to different history of exposure

to TB.

Other CD209 regions, particularly exon 4, have been

investigated in the context of TB. Several studies suggest

no significant differences between TB patients and controls

[14, 15, 17, 29], in accordance with our results (Table 4)

that indicate a low variability of exon 4 and confirm the

predominance of the allele with 7 repeats and 7/7 geno-

types in both patients and controls, ruling out an involve-

ment with the disease [14, 15, 17].

In contrast, the exon 4 of CD209L gene showed high

variability among our samples and some associations with

TB were observed. Allele with 5 repeats and the hetero-

zygous 6/5 repeats genotype conferred protection to TB

development, while the allele with 9 repeats associated

with increased susceptibility. Barreiro et al. [15], showed

higher frequencies of 6 and 7 alleles, and 7/7 and 7/6

genotypes in South African TB patients, but none of the

CD209L neck-region variants or genotypes seems to

influence the individual susceptibility to TB development.

To our knowledge, our and Barreiro et al. [15], are the

only two studies that investigated the possible involvement

of CD209L repeats in TB; nonetheless, CD209L neck

region polymorphism has been already associated with

other infections. Interestingly, Xu et al. [33], suggested an

association between the 9 repeats allele and susceptibility

to HIV infection and higher viral loads, compared with the

alleles with 5 repeats. The increased number of tandem-

repeats in CD209L neck region was shown to increase the

receptor stability and to allow a more efficient host-path-

ogen interaction [34, 35]. In the context of TB infection,

the presence of 9 repeats alleles could facilitate M. tuber-

culosis entry in the host cell, where it could escape the

action of lysosomal enzymes, multiply and affect new cells

[9].

Mol Biol Rep

123

Although our results reveal the association of 6/5

genotype with TB and a tendency to association of alleles

with 4, 5 and 6 tandem-repeats, it is not yet clear what the

effect of this variation in receptor function. We hypothe-

size that receptors with few tandem-repeats may disrupt the

interaction between the host and the pathogen receptor,

leading to prevention of M. tuberculosis entry, thus more

refined studies are needed to confirm our hypothesis.

The study of CD209 and CD209L genes in the context

of TB is important to clarify the roles of genetic poly-

morphisms in the development of the disease. Our results

are in favor of a possible involvement of both genes in TB

development, but other replica studies in different popu-

lations and functional studies are needed to confirm and

reinforce our findings.

Acknowledgments We thank the Laboratory of Immunopathology

Keizo Asami, the Department of Genetics, Federal University of

Pernambuco, the Graduate Program in Genetics for supporting

physical and scientific, as well as FACEPE and CNPq, for financial

support. We thank the Program for Technological Development in

Tools for Health-PDTIS/FIOCRUZ for use of its facilities.

Conflict of interest None of the authors has any potential financial

conflict of interest related to this manuscript.

References

1. World Health Organization (2010) Global tuberculosis control:

surveillance, planning, financing. WHO report. WHO, Geneva

2. Hernandez-Pando R, Orozco H, Aguilar D (2009) Factors that

deregulate the protective immune response in tuberculosis. Arch

Immunol Ther Exp 57:355–367. doi:10.1007/s00005-009-0042-9

3. Tailleux L, Pham-Thi N, Bergeron-Lafaurie A et al (2005) DC-

SIGN induction in alveolar macrophages defines privileged target

host cells for Mycobacteria in patients with tuberculosis. PLoS

Med 2(12):e381. doi:10.1371/journal.pmed.0020381

4. Khoo US, Chan KYK, Chan VSF, Lin CLS (2008) DC-SIGN and

L-SIGN: the SIGNs for infection. J Mol Med 86:861–874. doi:10.

1007/s00109-008-0350-2

5. Geijtenbeek TB, Torensma R, van Vliet SJ et al (2000) Identi-

fication of DC-SIGN, a novel dendritic cell-specific ICAM-3

receptor that supports primary immune responses. Cell

100(5):575–585. doi:10.1007/s00109-008-0350-2

6. Soilleux EJ, Barten R, Trowsdale J (2000) Cutting edge: DC-

SIGN; a related gene, DC-SIGNR; and CD23 form a cluster on

19p13. J Immunol 165:2937–2942

7. Soilleux EJ, Morris LS, Leslie G et al (2002) Constitutive and

induced expression of DC-SIGN on dendritic cell and macro-

phage subpopulations in situ and in vitro. J Leukoc Biol

71(3):445–457

8. Bashirova AA, Geijtenbeek TB, van Duijnhoven GC et al (2001)

A dendritic cell-specific intercellular adhesion molecule 3-grab-

bing nonintegrin (DC-SIGN)-related protein is highly expressed

on human liver sinusoidal endothelial cells and promotes HIV-1

infection. J Exp Med 193(6):671–678

9. Zhou T, Chen Y, Hao L, Zhang Y (2006) DC-SIGN and immu-

noregulation. Cell Mol Immunol 3(4):279–283

10. Boily-Larouche G, Zijenah LS, Mbizvo M, Ward BJ, Roger M

(2007) DC-SIGN and DC-SIGNR genetic diversity among dif-

ferent ethnic populations: potential implications for pathogen

recognition and disease susceptibility. Hum Immunol

68(6):523–530. doi:10.1016/j.humimm.2007.02.002

11. Selvaraj P, Alagarasu K, Swaminathan S, Harishankar M, Na-

rendran G (2009) CD209 gene polymorphisms in South Indian

HIV and HIV-TB patients. Infect Genet Evol 9(2):256–262.

doi:10.1016/j.meegid.2008.12.003

12. World Health Organization (1998) Tuberculosis handbook.

WHO, Geneva

13. Alves-Silva J, Da Silva Santos M, Guimaraes PE (2000) The

ancestry of Brazilian mtDNA lineages. Am J Hum Genet

67(2):444–461. doi:10.1086/303004

14. Alagarasu K, Selvaraj P, Swaminathan S, Raghavan S, Narendran

G, Narayanan PR (2009) CCR2, MCP-1, SDF-1a & DC-SIGN

gene polymorphisms in HIV-1 infected patients with & without

tuberculosis. Indian J Med Res 130:444–450

15. Barreiro LB, Neyrolles O, Babb CL et al (2007) Length variation

of DC-SIGN and L-SIGN neck-region has no impact tuberculosis

susceptibility. Hum Immunol 68(2):106–112

16. Rathore A, Chatterjee A, Sivarama P, Yamamoto N, Dhole TN

(2008) Role of homozygous DC-SIGNR 5/5 tandem repeat

polymorphism in HIV-1 exposed seronegative north Indian

individuals. J Clin Immunol 28(1):50–57

17. Ben-Ali M, Barreiro LB, Chabbou A et al (2007) Promoter and

neck region length variation of DC-SIGN is not associated with

susceptibility to tuberculosis in Tunisian patients. Hum Immunol

68(11):908–912

18. Excoffier L, Laval G, Schneider S (2005) Arlequin (version 3.0):

an integrated software package for population genetics data

analysis. Evol Bioinform Online 1:47–50

19. R Development Core Team (2010) R: a language and environ-

ment for statistical computing. Viena

20. Sole X, Guino E, Valls J, Iniesta R, Moreno V (2006) SNPStats: a

web tool for the analysis of association studies. Bioinformatics

22(15):1928–1929

21. Liu H, Yu W, Liou LY, Rice AP (2003) Isolation and charac-

terization of the human DC-SIGN and DC-SIGNR promoters.

Gene 313:149–159

22. Sakuntabhai A, Turbpaiboon C, Casademont I et al (2005) A

variant in the CD209 promoter is associated with severity of

dengue disease. Nat Genet 37(5):507–513

23. Sainz J, Segura-Catena J, Jura do M (2010) Association between

genetic polymorphism in the promotor region off CD209 and

propensity to develop invasive pulmonary aspergillosis. Methods

Find Exp Clin Pharmacol 32(Suppl A):9–13

24. Kashima S, Rodrigues ES, Azevedo R et al (2009) DC-SIGN

(CD209) gene promoter polymorphisms in a Brazilian population

and their association with human T-cell lymphotropic virus type

1 infection. J Gen Virol 90(Pt 4):927–934

25. Barkhash AV, Perelygin AA, Babenko VN, Brinton MA, Voe-

voda MI (2012) Single nucleotide polymorphism in the promoter

region of the CD209 gene is associated with human predisposi-

tion to severe forms of tick-borne encephalitis. Antiviral Res

93(1):64–68

26. Mezger M, Steffens M, Semmler C et al (2008) Investigation of

promoter variations in dendritic cell-specific ICAM3-grabbing

non-integrin (DC-SIGN) (CD209) and their relevance for human

cytomegalovirus reactivation and disease after allogeneic stem-

cell transplantation. Clin Microbiol Infect 14(3):228–234

27. Vannberg FO, Chapman SJ, Khor CC et al (2008) CD209 genetic

polymorphism and tuberculosis disease. PLoS One 3(1):e1388

28. Barreiro LB, Neyrolles O, Babb CL et al (2006) Promoter vari-

ation in the DC-SIGN-encoding gene CD209 is associated with

tuberculosis. PLoS Med 3(2):e20

29. Gomez LM, Anaya JM, Sierra-Filardi E, Cadena J, Corbi A,

Martın J (2006) Analysis of DC-SIGN (CD209) functional vari-

ants in patients with tuberculosis. Hum Immunol 67(10):808–811

Mol Biol Rep

123

30. Zheng J, Zhou Y, Qin L et al (2011) Relationship between

polymorphism of DC-SIGN (CD209) gene and the susceptibility

to pulmonary tuberculosis in an eastern Chinese population. Hum

Immunol 72(2):183–186

31. Kobayashi K, Yuliwulandari R, Yanai H, Lien LT, Hang NT,

Hijikata M, Keicho N, Takunaga K (2011) Association of CD209

polymorphisms with tuberculosis in an Indonesian population.

Hum Immunol 72(9):741–745

32. The International HapMap Consortium (2005) A haplotype map

of the human genome. Nature 437:1299–1320

33. Xu L, Li Q, Ye H et al (2010) The nine-repeat DC-SIGNR iso-

form is associated with increased HIV-RNA loads and HIV

sexual transmission. J Clin Immunol 30(3):402–407

34. Guo Y, Atkinson CE, Taylor ME, Drickamer K (2006) All but the

shortest polymorphic forms of the viral receptor DC-SIGNR

assemble into homo- and heterotetramers. J Biol Chem

281(24):16794–16798

35. Gramberg T, Zhu T, Chaipan C et al (2006) Impact of poly-

morphisms in the DC-SIGNR neck domain on the interaction with

pathogens. Virology 347:354–363

Mol Biol Rep

123