Kaohsiung Medical University Institutional Repository:Item 310902000/11290

Solute Carrier Family 11 Member A1 Gene Polymorphisms

in Reactive Arthritis

YI-JING CHEN,

CHIA-HUI LIN,

TSAN-TENG OU,

CHENG-CHIN WU,

WEN-CHAN TSAI,

HONG-WEN LIU,

and JENG-HSIEN YEN

Received June 16, 2006; accepted September 26, 2006 Published online: 9 January 2007

To investigate the role of SLC 11A1 polymorphisms in the

devel-opment of reactive arthritis, 91 patients with reactive arthritis and

163 healthy controls were enrolled in this study. The SLC 11A1

polymorphisms were determined by the method of polymerase

chain reaction/restriction fragment length polymorphism. The

genotype distributions of SLC 11A1 274, 823, 1703, and 1729

+

55 del 4 were significantly different between the patients with

reactive arthritis and controls. The genotype frequency of SLC

11A1 274C/C was significantly decreased in the patients with

reactive arthritis when compared with that of the controls. In

contrast, the SLC 11A1 274C/T showed a significant association

with reactive arthritis. The patients with reactive arthritis have

a significantly higher frequency of SLC 11A1 823C/C than the

controls. However, SLC 11A1 823T/T was resistant to the

devel-opment of reactive arthritis. The allele frequencies of SLC 11A1

274T and 823C were significantly increased in the patients with

reactive arthritis in comparison with those of the controls,

inde-pendent of HLA-B27. On the contrary, the allele frequencies of

SLC 11A1 274C and 823T were significantly decreased in the

pa-tients with reactive arthritis. The estimated haplotype frequency

of SLC 11A1 274C 823T 1703G 1729

+ 55del 4 TGTG + was

significantly decreased in the patients with reactive arthritis

when compared with that of the controls. In contrast, the

es-timated haplotype frequency of SLC 11A1 274T 823C 1703G

1729

+ 55 del 4 TGTG + was significantly increased in the

patients with reactive arthritis. This study shows that the SLC

1_{Graduate Institute of Medicine, College of Medicine, Kaohsiung}

Med-ical University, Kaohsiung, Taiwan.

2_{Division of Rheumatology, Department of Internal Medicine,}

Kaohsi-ung Medical University Hospital, KaohsiKaohsi-ung City, Taiwan.

3_{Faculty of Medicine, College of Medicine, Kaohsiung Medical}

Univer-sity, Kaohsiung, Taiwan.

4_{Division of Rheumatology, Department of Internal Medicine,}

Kaoh-siung Medical University Hospital, 100 Zihyou 1st Road, KaohKaoh-siung City 807, Taiwan.

5_{To whom correspondence should be addressed e-mail: jehsye@}

kmu.edu.tw.

11A1 274T and 823C alleles are associated with susceptibility to

reactive arthritis independently of HLA-B27 in Taiwan. The SLC

11A1 274T 823C 1703G 1729

+ 55 del 4 TGTG + haplotype is

associated with the development of reactive arthritis in Taiwan.

In contrast, the SLC 11A1 274C 823T 1703G 1729

+ 55 del 4

TGTG

+ haplotype may be a protective factor.

KEY WORDS: Reactive arthritis; SLC 11A1; NRAMP1; chlamydia.

INTRODUCTION

Reactive arthritis is a form of arthritis occurring after

genitourinary or gastrointestinal tract infections caused

by certain pathogens including Salmonella, Shigella,

Yersinia, Campylobacter, and Chlamydia trachomatis.

Several other pathogens may also be involved. Reactive

arthritis typically occurs acutely 1–4 weeks after

infec-tion, and the genitourinary tract infection is frequently

asymptomatic. Reactive arthritis is usually asymmetric

and oligoarticular. Most patients with reactive arthritis run

a self-limited course. However, about 15% of patients

con-tinue to have chronic, destructive, and disabling arthritis or

enthesitis. In addition, some patients have extra-articular

manifestations including keratoderma blennorhagicum,

circinate balanitis, uveitis, and aortitis (

1

,

2

).

Chlamydia trachomatis is a major pathogen of

gen-itourinary tract infection in reactive arthritis. However,

only a minor proportion of patients with Chlamydia

tra-chomatis infection develop reactive arthritis (3

), the

rea-son for which is still unknown. Genetic factors may play

a role in the development of this disease. HLA-B27 is

related to the increased disease susceptibility and

influ-ences disease severity and expression (

4

–

6

). Tuokko et al.

also showed that TNFc1 might be a new susceptibility

marker for reactive arthritis independent of HLA-B27 (

7

).

Our recent study also revealed associations of cytochrome

p450 1A1 and manganese superoxide dismutase genes

with reactive arthritis following Chlamydial infection (

8

).

Solute carrier family 11 member A1 (SLC 11A1), also

called natural resistance-associated macrophage protein 1,

is involved in killing intracellular pathogens (

9

).

Chlamy-dia trachomatis is an intracellular pathogen. Therefore,

SLC 11A1 may be related to the reactive arthritis

fol-lowing Chlamydial infection. The SLC 11A1

polymor-phisms may have different effects on the killing of these

pathogens, and then influence the development and

sever-ity of reactive arthritis.

A study on the association of SLC 11A1 with

reac-tive arthritis is still unavailable in the literature. This is

the first study of SLC 11A1 polymorphisms in reactive

arthritis, the purpose of which is to investigate the role of

SLC 11A1 polymorphisms in the development and clinical

manifestations of reactive arthritis.

MATERIALS AND METHODS

The diagnosis of reactive arthritis is made according

to the criteria proposed in the Third International

Work-shop on Reactive Arthritis (

10

). Only reactive arthritis

patients with active Chlamydial infection were enrolled

in this study, as confirmed by an indirect

immunoperoxi-dase assay with either antichlamydial IgG-Ab

≥ 1:128 or

both antichlamydial IgG-Ab

≥ 1:64 and IgA-Ab ≥ 1:16

(IPAzyme Chlamydia kit, Savyon Diagnostics Ltd.,

Is-arel). Ninety-one patients (48 males, 43 females) with

reactive arthritis following urogenital Chlamydial

infec-tion and 163 age- and sex-matched healthy controls (83

males, 80 females) were enrolled in this study. All the

patients and controls are Taiwanese. This study was

ap-proved by the Institutional Review Board of Kaohsiung

Medical University Hospital.

SLC 11A1 polymorphisms were determined by the

polymerase chain reaction/ restriction fragment length

polymorphism method, including 274 C/T (at codon 66

in exon 3), 469

+ 14 G/C (at nucleotide + 14 of intron 4),

577–18 G/A (at nucleotide

− 18 of intron 5), 823 C/T (at

codon 249 or nucleotide 823 in exon 8), A318V (at codon

318 in exon 9; C or T at nucleotide 1029), 1465–85 G/A

(at nucleotide

− 85 of intron 13), D543N (at codon 543

in exon 15; G or A at nucleotide 1703), and 1729

+ 55 del

4 (deletion of TGTG in the 3

-UTR; 55 nucleotides 3

to

the last codon in exon 15). The sequences of primers and

restriction enzymes are given in Table

I

.

The chi-square test or Fisher’s exact test was used for

statistical analysis. Odds ratio (OR) and its 95%

confi-dence interval (CI) were calculated by the SPSS statistical

program. The Haldane method was used to calculate OR

if the value of any cell is 0 (

11

). The delta values (
)

were calculated to evaluate the linkage disequilibrium.

To calculate the estimated haplotype frequencies, the EH

program was used (Web Resources of Genetic Linkage

Analysis). The Mantel–Haenszel test was used in a

statis-tic with stratification.

RESULTS

The genotypes of SLC 11A1 in the controls appeared to

be in Hardy–Weinberg equilibrium. The genotype

distri-butions of SLC 11A1 274C/T, 823C/T, 1703G/A (D543N),

and 1729–55 del4 TGTG/-polymorphisms were

signif-icantly different between the patients with

Chlamydia-induced reactive arthritis and the healthy controls

(Table

II

). The patients with reactive arthritis have a

significantly lower genotype frequency of SLC 11A1

Table I. The Sequences of Primers and Restriction Enzymes in Determining SLC 11A1 Polymorphisms

SLC 11A1 Primers Restriction enzymes

274 C/T 5-TGCCACCATCCCTATACCCAG-3 Mnl I 5-CTTCAACACTTAGCCTGGTCAC-3 469+ 14G/C 5-TCTCTGGCTGAAGGCCTCTCC-3 Apa I 5-TGTGCTATCAGTTTGAGCCTC-3 577–18G/A 5-CTGGACCAGGCTGGGCTGAC-3 Msp I 5-CCACCACTCCCCTATGAGGTG-3 823 C/T 5-CTTGTCCTGACCAGGCTCCT-3 Nar I 5-CATGGCTCCGACTGAGTGAG-3

A318V (1029C/T) 5-TCCCTTTGATCTTCGTAGTCTC-3 BstU I 5-GGCTTACAGGACATGAGTAC-3

1465–85G/A 5-GCAAGTTGAGGAGCCAAGAC-3 Bsr I

5-ACCTGCATCAACTCCTCTTC-3

D543N (1703G/A) 5-GCATCTCCCCAATTCATGGT-3 Ava II 5-AACTGTCCCACTCTATCCTG-3

1729+ 55 del 4 TGTG + /del 5-GCATCTCCCCAATTCATGGT-3 BstF5 I 5-AACTGTCCCACTCTATCCTG-3

Table II. Genotype Frequencies of SLC 11A1 Polymorphisms in the Patients with Reactive Arthritis and Controls

SLC 11A1 genotypes

Reactive arthritis

n= 91 (%) Controls n= 163 (%)

p Value (for overall

genotype frequency)

p Value (for individual

genotype frequency) OR (95% CI) 274 C/C 69 (75.8)∗ 145 (88.9) 0.02 1 C/T 21 (23.1) 17 (10.4) 0.006 2.6 (1.3–5.2) T/T 1 (1.1) 1 (0.6) 469+ 14 G/G 72 (79.1) 144 (88.3) NS G/C 18 (19.8) 17 (10.4) C/C 1 (1.1) 2 (1.2) 577–18 G/G 84 (92.3) 148 (90.8) NS G/A 6 (6.6) 14 (8.6) A/A 1 (0) 1 (0.6) 823 C/C 76 (83.5)# _{106 (65.0) 0.002 1} C/T 15 (16.5) 45 (27.6) T/T 0 (0) 12 (7.4) 0.004 0.06 (0.01–0.4) 1029 (A318V) C/C (A/A) 91 (100.0) 161 (98.8) NS C/T (A/V) 0 (0) 2 (1.2) T/T (V/V) 0 (0) 0 (0) 1465-85 G/G 41 (45.1) 76 (46.6) NS G/A 47 (51.6) 78 (47.9) A/A 3 (3.3) 9 (5.5) 1703 (D543N) G/G (D/D) 69 (75.8) 108 (66.3) 0.04 G/A (D/N) 19 (20.9) 54 (33.1) A/A (N/N) 3 (3.3) 1 (0.6) 1729+ 55del 4 TGTG+ / + 69 (75.8) 106 (65.0) 0.03 TGTG+ /del 19 (20.9) 56 (34.4) TGTG del/del 3 (3.3) 1 (0.6)

Note. NS, not significant.

∗_{Reactive arthritis versus Controls: p= 0.006, OR = 0.4, 95% CI = 0.2–0.8.}

#_{Reactive arthritis versus Controls: p= 0.002, OR = 2.7, 95% CI = 1.4–5.2.}

274C/C than the controls (p

= 0.006, OR = 0.4, 95%

CI

= 0.2–0.8). In comparison with SLC 11A1 274 C/C,

the patients with reactive arthritis have a significantly

higher frequency of SLC 11A1 274 C/T than the controls

(p

= 0.006, OR = 2.6, 95% CI = 1.3–5.2). We also

found that the genotype frequency of SLC 11A1 823C/C

was significantly increased in the patients with

reac-tive arthritis when compared with that of the controls (

p

= 0.002, OR = 2.7, 95% CI = 1.4–5.2). In comparison

with SLC 11A1 823 C/C, the patients with reactive

arthri-tis have a significantly lower genotype frequency of SLC

11A1 823T/T than the controls ( p

= 0.004, OR = 0.06,

95% CI

= 0.01–0.4).

The allele frequency of SLC 11A1 274T was

signifi-cantly higher in patients with reactive arthritis than that

of the controls (Table

III

, p

= 0.008, OR = 2.3, 95%

CI

= 1.2–4.4). A similar finding could also be found in

SLC 11A1 823C. The patients with reactive arthritis have

a significantly higher allele frequency of SLC 11A1 823C

than the controls (Table

III

, p < 0.001, OR

= 3.0, 95%

CI

= 1.7–5.4). However, the patients with reactive

arthri-tis have significantly lower allele frequencies of SLC 11A1

274C and 823T than the controls (p

= 0.008, OR = 0.4,

95% CI

= 0.2–0.8, and p < 0.001, OR = 0.3, 95%

CI

= 0.2–0.6, respectively).

The haplotype frequency of SLC 11A1 274C 823T

1703G 1729

+ 55 del 4 TGTG + was significantly

de-creased in the patients with reactive arthritis when

com-pared with that of the controls (Table

IV

, p < 0.001,

OR

= 0.02, 95% CI = 0–0.2). In contrast, the

pa-tients with reactive arthritis have a significantly higher

haplotype frequency of SLC 11A1 274T 823C 1703G

1729

+ 55 del 4 TGTG + than the controls (p = 0.004,

OR

= 2.4, 95% CI = 1.2–4.6).

This study also revealed that HLA-B27 was associated

with the development of reactive arthritis (positive

HLA-B27: patients vs. controls

= 34.1% vs. 6.7%, p < 0.001,

Table III. Allele Frequencies of SLC 11A1 Polymorphisms in the Patients with Reactive Arthritis and Controls SLC 11A1 allele frequencies Reactive arthritis 2n= 182 (%) Controls 2n= 326 (%) p OR (95% CI) 274 C 159 (87.4) 307 (94.2) 0.008 0.4 (0.2–0.8) T 23 (12.6) 19 (5.8) 0.008 2.3 (1.2–4.4) 469+ 14 G 162 (89.0) 305 (93.6) NS C 20 (11.0) 21 (6.4) 577-18 G 174 (95.6) 310 (95.1) NS A 8 (4.4) 16 (4.9) 823 C 167 (91.8) 257 (78.8) <0.001 3.0 (1.7–5.4) T 15 (8.2) 69 (21.2) <0.001 0.3 (0.2–0.6) 1029 C (318 A) 182 (100.0) 324 (99.4) NS T (318 V) 0 (0) 2 (0.6) 1465–85 G 129 (70.9) 230 (70.6) NS A 53 (29.1) 96 (29.4) 1703 G (543D) 157 (86.3) 270 (82.8) NS A (543N) 25 (13.7) 56 (17.2) 1729+ 55 del 4 TGTG+ 157 (86.3) 268 (82.2) NS TGTG del 25 (13.7) 58 (17.8)

Note. OR, odds ratio.

clarify the role of SLC 11A1 in the pathogenesis of

re-active arthritis, the allele frequencies of SLC 11A1

poly-morphisms were stratified with HLA-B27 (Table

V

). The

Mantel–Haenszel test was used for statistical analysis. It

showed that the association of SLC 11A1 274T with

reac-tive arthritis was independent of HLA-B27 (p

= 0.03,

adjusted OR

= 2.2, 95% CI = 1.1–4.4). The

asso-ciation between SLC 11A1 823C and reactive arthritis

was also independent of HLA-B27 (p

= 0.001, adjusted

OR

= 3.1, 95% CI = 1.6–5.9). In contrast, SLC 11A1

274C and 823T resist the development of reactive arthritis

independently of HLA-B27 (p

= 0.03, OR = 0.5, 95%

CI

= 0.2–0.9, and p = 0.001, OR = 0.3, 95% CI = 0.2–

0.6, respectively).

DISCUSSION

This study has demonstrated that SLC 11A1 274T and

823C are associated with the development of reactive

arthritis. The SLC 11A1 274T 823C 1703G 1729

+ 55

del 4 TGTG

+ haplotype is related to susceptibility to

reactive arthritis, while the SLC 11A1 274C 823T 1703G

1729

+ 55 del 4 TGTG + haplotype is a protective factor.

Table IV. Estimated Haplotype Frequencies of SLC 11A1 Polymorphisms in the Patients with Reactive Arthritis and Controls Haplotype of SLC 11A1 Reactive arthritis Controls p OR (95% CI) 274C 823C 1703G 1729+ 55 del 4 TGTG + 0.743 0.670 NS

274C 823C 1703A 1729+ 55 del 4 TGTG del 0.058 0.060 NS

274C 823T 1703G 1729+ 55 del 4 TGTG + 0 0.099 <0.001 0.02 (0–0.2) 274C 823T 1703G 1729+ 55 del 4 TGTG del 0 0.006 NS

274C 823T 1703A 1729+ 55 del 4 TGTG del 0.078 0.101 NS

274T 823C 1703G 1729+ 55 del 4 TGTG + 0.114 0.05 0.004 2.4 (1.2–4.6) 274T 823C 1703A 1729+ 55 del 4 TGTG del 0.002 0.007 NS

274T 823T 1703G 1729+ 55 del 4 TGTG + 0 0.006 NS 274T 823T 1703A 1729+ 55 del 4 TGTG del 0.005 0 NS

Table V. Allele Frequencies of SLC 11A1 Polymorphisms Stratified by HLA-B27 in the Patients with Reactive Arthritis and Controls

HLA-B27(+ ) HLA-B27(− )

Reactive A Controls Reactive A Controls

SLC 11A1 2n= 62(%) 2n= 22(%) 2n= 120(%) 2n= 304(%) P Adjusted OR (95% CI)

274T 12 (19.4) 0 (0) 11 (9.2) 19 (6.3) 0.03 2.2 (1.1–4.4)

823C 56 (90.3) 18 (81.8) 111 (92.5) 239 (78.6) 0.001 3.1 (1.6–5.9) 274C 50 (80.6) 22 (100) 109 (90.8) 285 (93.8) 0.03 0.5 (0.2–0.9) 823T 6 (9.7) 4 (18.2) 9 (7.5) 65 (21.4) 0.001 0.3 (0.2–0.6)

Note. The Mantel–Haenszel test was used for statistical analysis. Reactive A, reactive arthritis.

SLC 11A1 has pleiotropic effects on macrophage

func-tions, including antimicrobial activity, tumoricidal

activ-ity, and upregulation of chemokine/cytokine gene, tumor

necrosis factor α (TNFα), interleukin 1 β(IL-1β),

in-ducible nitric oxide synthase (iNOS), as well as major

histocompatibility complex expression (

12

–

15

). These

ef-fects are involved in resistance to infection and may also

be involved in induction and maintenance of autoimmune

disease (

12

). Our previous study also showed that SLC

11A1 was related to the susceptibility and clinical

mani-festation of rheumatoid arthritis (

16

).

SLC 11A1 is located on chromosome 2q35. SLC 11A1

expression is primarily in tertiary granules of

polymor-phonuclear cells and macrophages. Then, it is recruited

from tertiary granules to the phagosomal membrane on

phagocytosis (

17

). SLC 11A1 is a proton/divalent cationic

(Fe

, Zn

, Co

, Mn

, and Mg

) antiporter (

18

–

20

), which fluxes divalent cations in either direction

de-pending on the PH on either side of the membrane (

21

).

In late endosomes/lysosomes, SLC 11A1 delivers

diva-lent cations from the cytosole to this acidic

compart-ment. The Fenton reaction uses ferrous iron to

gener-ate toxic hydroxyl radicals, which are associgener-ated with

killing intracellular pathogens (

9

). The expression of

iNOS and generation of toxic NO may be influenced by

the polymorphisms of SLC 11A1 (

22

,

23

). Mutation of

SLC 11A1 also impairs phagosomal acidification, which

is related to intracellular infection (

24

). Vidal showed

that a mutation at amino acid 169 of SLC 11A1 made

mice more susceptible to infection by many pathogens

(

25

). SLC 11A1 polymorphisms are also related to many

human infectious diseases, including pulmonary

tubercu-losis (TB), leprosy, human immunodeficiency virus

in-fection, visceral leishmaniasis, and meningococal

menin-gitis (

26

–

30

). The associations of SLC 11A1

polymor-phisms with TB infection are still controversial. However,

the polymorphisms at different sites may be associated

with TB susceptibility or resistance in different ethnic

groups (

31

).

In addition to killing intracellular pathogens, SLC 11A1

also upregulates the expression of TNFα and IL-1β by

macrophages. TNFα and IL-1β play important roles in

in-flammation and tissue destruction of inflammatory

arthri-tis including reactive arthriarthri-tis (

32

). Therefore, SLC 11A1

may be related to the pathogenesis of reactive arthritis.

Smit showed that SLC 11A1 might provide a link between

the genetic background, the bacterial environmental, and

the development of allergic diseases (

33

). A similar

con-dition may also be present in reactive arthritis.

This study has demonstrated the associations between

SLC 11A1 polymorphisms and development of reactive

arthritis. SLC 11A1 274T and 823C are related to

suscep-tibility to reactive arthritis independently of HLA-B27. On

the contrary, SLC 11A1 274C and 823T prevent the

de-velopment of reactive arthritis. A linkage disequilibrium

is present between SLC 11A1 1703G and 1729

+ 55 del 4

TGTG

+ (p < 0.0001,
= 0.18, data not shown). The

SLC 11A1 274T 823C 1703G 1729

+ 55 del 4 TGTG +

haplotype is a precipitating factor for reactive arthritis.

In contrast, the SLC 11A1 274C 823T 1703G 1729

+ 55

del 4 TGTG

+ haplotype is a protective factor. This study

also reveals that the SLC 11A1 274C/T, 823C/T, 1703G/A,

and 1729

+ 55 del 4 TGTG + /del polymorphisms are not

related to clinical manifestations of reactive arthritis

in-cluding conjunctivitis, uveitis, oral ulcer, circinate

balani-tis, keratoderma blenorrhagica, and enthesopathy (data

not shown).

The associations of SLC 11A1 with immune-mediated

diseases, including rheumatoid arthritis, juvenile

rheuma-toid arthritis, type 1 diabetes, multiple sclerosis, and

Crohn’s disease, have also been demonstrated (

34

–

39

).

In summary, SLC 11A1 274T and 823C are risk factors

for the development of reactive arthritis independent of

HLA-B27. The SLC 11A1 274T 823C 1703G 1729

+ 55

del 4 TGTG

+ haplotype is associated with susceptibility

to reactive arthritis in Taiwan, and the SLC 11A1 274C

823T 1703G 1729

+ 55 del 4 TGTG + haplotype is

as-sociated with resistance to reactive arthritis.

ACKNOWLEDGMENTS

This study was supported by grants from the National

Science Council of Taiwan (NSC 93-2314-B-037-034)

and Kaohsiung Medical University.

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