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Indian J Med Res 123, June 2006, pp 815-820
Interleukin-1 receptor antagonist gene polymorphism in patients
with rheumatoid arthritis in India
S. Grover, S. Tandon, R. Misra & A. Aggarwal
Department of Immunology, Sanjay Gandhi Postgraduate Institute of Medical Sciences
Lucknow, India
Received August 12, 2005
Background & objectives: Interleukin-1 receptor antagonist (IL-1Ra) is a naturally occurring
anti-inflammatory molecule that blocks action of IL-1. Polymorphism in IL-1Ra gene intron 2
results in differences in production of IL-1Ra. These polymorphisms are reportedly associated
with autoimmune disease susceptibility in different studies. However, such data are lacking
from India. We undertook this study to examine the IL-1Ra polymorphism as a susceptibility
marker in patients with rheumatoid arthritis (RA).
Methods: DNA samples from 107 patients with RA and 111 healthy controls were used to study
genotypes of the IL-1RA gene by PCR. Allelic frequencies and carriage rates were calculated
and compared in both the groups.
Results: Among the 107 patients with RA, 93 were females and 75 per cent were seropositive for
rheumatoid factor. The frequencies of IL-1RA alleles in controls were as follows: Allele 1 (IL1RN*1) was 83.33 per cent, IL-1RA allele 2 (IL-1RN*2) was 16.21 per cent and allele 3 (0.46%).
In RA patients the allele frequencies were 84.11 per cent for IL-1RN*1, 14.95 per cent for IL1RN*2, 0.47 per cent each for IL-1RN*3 and IL-1RN*4. There was no difference in frequency of
different alleles between the two groups. However, homozygosity for allele 2 was more frequent
in controls (9.91%) as compared to patients (4.67%).
Interpretation & conclusion: Our findings indicated that IL-1RA polymorphism was not a
susceptibility marker in RA nor did it show any association with seropositivity, Sjögren’s
syndrome or subcutaneous nodules. Further studies with large sample need to be done to
confirm these findings.
Key words Cytokine antagonist - interleukin-1 receptor - rheumatoid arthritis - Sjögren’s syndrome
including India 2. RA is a complex multi-factorial
disease in which genetic susceptibility affects
disease occurrence and severity. HLA class II DR4
alleles are known to be associated with more severe
disease 3,4 , however, HLA complex represents only
Rheumatoid arthritis (RA) is a chronic systemic
inflammatory disease characterized by synovial
inflammation, bone and cartilage destruction with
a variety of extra-articular features. It affects
approximately 1 per cent of world population 1
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30-50 per cent of the total genetic susceptibility5.
Thus, non-HLA genes including cytokine gene
polymorphism may be important in RA.
Interleukin-1 (IL-1) plays an important role in
mediating joint inflammation and destruction in RA.
The IL-1 gene family contains three related genes,
IL-1A, IL-1B and IL-1RN, which encode the proinflammatory cytokines IL-1α, IL-1β and their
naturally occurring antagonist, interleukin-1 receptor
antagonist (IL-1Ra), respectively. Il-1Ra acts by
competitive blockade of IL-1 receptor without
affecting downstream signaling 6. IL-1Ra knockout
mice spontaneously develop inflammatory arthritis
resembling RA 7, and exogenous administration of
IL-1Ra prevents bone damage in animal models 8
and joint erosion in patients of rheumatoid arthritis 9.
Recombinant IL-1Ra (Anakinra) is now an
established therapeutic agent in the management
of RA. The gene for IL-1Ra (IL-1RN) exists in 5
allelic variants corresponding to 2,3,4,5 and 6 copies
of 86-base pair sequence repeat located in intron
2 10 . Since three potential protein binding sites are
located in this 86-bp sequence, the number of
repeats may influence gene transcription and protein
production. Moreover, one allele IL-1RN*2 has
been associated as a risk and/or severity factor
with various autoimmune diseases like systemic
lupus erythematosus 11 , Sjögren’s syndrome 12 ,
juvenile idiopathic arthritis13, ankylosing spondylitis14
and inflammatory bowel disease 15 . The data
regarding association of allele 2 with RA are
conflicting and there are no data from India. Thus,
we examined the VNTR (variable number of tandem
repeats) polymorphism of IL-1RN in patients with
RA to determine its association with susceptibility
and severity of disease.
Material & Methods
This retrospective study was conducted at the
Department of Immunology, Sanjay Gandhi
Postgraduate Institute of Medical Sciences,
Lucknow, in north India. The sample size calculation
was based on the following assumptions: prevalence
of allele 2 of 22 per cent (20-27%), significance
level of 0.05 per cent, power of study 80 per cent,
and a reported relative risk for allele 2 in
autoimmune diseases of 2.2. The sample size was
calculated to be 97 in both controls and patients
groups. Finally, stored DNA samples collected
between June 2003 and April 2004 for another study
from 107 patients with RA [diagnosed based on
American Rheumatism Association (ACR) 1987
criteria 16] and from 111 unrelated healthy individuals
(hospital employee and unrelated attendants of
patients) from similar ethnic background were
analyzed for IL-1RN gene polymorphism. Genomic
DNA had been isolated from EDTA anti-coagulated
blood using salting out technique 17 .
Data regarding age of onset, rheumatoid factor
(RF) seropositivity, extra-articular features
(subcutaneous nodules, secondary Sjögren’s
syndrome, vasculitis and interstitial disease) were
collected from hospital records. Patients having
RF seropositivity and/or extra-articular features
were classified as having severe disease.
PCR for IL-1RN VNTR polymorphism 10 : Genomic
DNA (1 µg) was amplified in 20 ml reaction
volume using Taq polymerase (0.5 units, Invitrogen,
USA), dNTP (200 µM, Invitrogen, USA),
oligonucleotide primers (1 µM each of the two
primers, 5’-CTCAGCAACACTCCTAT -3’ and 5’TCGTGGTCTGCAGGTAA-3’ Genosys, India) and
PCR buffer 10X (100 mM Tris HCl, 500 mM KCl
and 15 mM magnesium chloride). Forty cycles with
each cycle having denaturation at 95°C for 30 sec,
annealing at 62°C for 30 sec and extension at 72°C
for 30 sec were run. Ethidium bromide-stained
2 per cent agarose gels were used to visualize the
PCR products and the product size was calculated
relative to 100-bp DNA ladder. The product size
of different alleles was as follows:I IL1RN*1 410
bp; II L1RN*2 240 bp; III IL1RN*3 500 bp; IV
IL1RN*4 325 bp; and V IL1RN*5 595 bp.
Statistical analysis: Allelic frequencies (number
of copies of a specific allele divided by the total
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GROVER et al: IL-1RA POLYMORPHISM IN RA
number of alleles in the group) and carriage rates
(number of individuals with at least one copy of a
specific allele divided by the total number of
individuals within the group) were calculated. The
inter-group comparison of frequencies was done
using chi square and Fisher’s exact test. Odds ratio
(OR) were calculated for disease susceptibility or
severity in carriers of specific alleles. The 95 per
cent confidence intervals (95% CI) for the OR
were also determined.
Results
Among 107 patients with RA there were 93
females. The mean age was 45 ± (23-71) yr and
mean age of onset of disease was 36.74 (17-58) yr.
Rheumatoid factor was present in 80
patients (75%), 39 patients (36.44%) had
extra-articular features with 24 having subcutaneous
nodules, 21 secondary Sjögren’s syndrome,
9 interstitial lung disease and 2 had vasculitis.
The frequencies of different alleles were similar
in controls and patients. Genotype frequencies
showed a trend towards lesser frequency of allele
II homozygosity in patients. No difference was seen
in the carriage rate of different alleles (Table I).
There was no difference in allele or genotype
frequency in patients with rheumatoid factor and
those without it, patients with and without
subcutaneous nodules or Sjögren’s syndrome
(Table II) and those with early (<40 yr; n=61) or
late age of onset (n=46). Patients with Sjögren’s
syndrome had a lower frequency (4.76%) of allele
2 as compared to those who were negative for it
(17.44%) but this difference was not statistically
significant.
Table I. Comparison of frequencies observed in RA patients and controls
IL-1RN
allele
RA (n =107)
no. (% )
Controls (n =111)
no. (% )
Odds ratio
(95% CI)
180(84.11)
185 (83.33)
1.06 (0.62-1.82)
32(14.95)
36 (16.21)
0.91 (0.52-1.57)
III
1(0.47)
1 (0.46)
IV
1(0.47)
0
0
0
I/I
78 (72.90)
85 (76.58)
0.82 (0.43-1.59)
I / II
22 (20.57)
14 (12.61)
0.79 (0.82-3.97)
I / III
1(0.93)
1 (0.90)
1.034 (0 –38.13)
I / IV
1(0.93)
0
0
0
5(4.67)
11(9.91)
0.45 (0.13-1.45)
I
102 (95.32)
100 (90.09)
1.87 (0.61-5.94)
II
27 (25.23)
25 (22.52)
1.16 (0.59-2.27)
III
1 (0.93)
1 (0.90)
1.034 (0 –38.13)
IV
1 (0.93)
0
0
0
I
II
V
Genotype:
I/V
II / II
Carriage rate:
V
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INDIAN J MED RES, JUNE 2006
Table II. Allele and genotype frequency in two groups of patients with RA
RF positive
(N=80)
RF negative
(N=27)
SS present
(N=21)
SS absent
(N=86)
SC nodules
present
(N=24)
SC nodules
absent
(N=83)
Allele frequency:
I
135 (84.3)
45 (83.3)
39 (92.85)
141(81.97)
42(87.5)
138 (83.13)
24 (15)
8 (14.8)
2 (4.76)
30(17.44)
5 (10.41)
27 (16.26)
III
0
1 (1.85)
0
1 (0.58)
0
1 (0.61)
IV
1 (0.6)
0
1 (2.38)
0
1(2.08)
0
0
0
0
0
0
0
59 (73.7)
19 (70.4)
18(85.71)
60(69.76)
19 (79.16)
59 (71.08)
16 (20)
6 (22.2)
2 (9.52)
20(23.25)
3 (12.5)
19 (22.89)
I / III
0
1 (3.7)
0
1 (1.16)
0
1(1.20)
I / IV
1 (1.25)
0
1(4.76)
0
1 (4.16)
0
0
0
0
0
0
0
4 (5)
1 (3.7)
0
5
1
4
II
V
Genotype frequency:
I/I
I / II
I/V
II / II
RF, rheumatoid factor; SS, Sjögren syndrome; SC-subcutaneous
None of the comparisons were significant
Value in parentheses are percentages
Discussion
Cytokines are key players in pathogenesis of
synovitis and subsequent joint damage in RA.
Difference in levels of various cytokines among
different individuals can be a possible explanation
for differences in disease susceptibility and severity.
These variations are mostly due to polymorphism
in the genes encoding for cytokines. IL-1, a cytokine
produced by monocytes mediates cartilage and
bone destruction in RA. IL-1α, IL-1β and their
naturally occurring antagonist, IL-1Ra are coded
by genes located within a 430KB region 18 . IL-1Ra
counteracts the action of IL-1 by binding to IL-1
receptor without activating it 6 .
No difference was found in allele frequency,
genotype frequency and carriage rate for IL-1Ra
between patients with RA and healthy controls.
There was a trend towards less frequency of
IL1RN*2 homozygosity in patients with RA.
Among our population, the 4 repeat (IL1RN*1,allele 1) and 2 repeat (IL-1RN*2,allele 2 )
were found in majority of patients and controls.
The frequency of IL1RN*1 allele was more
common in Indians than in French 12 , British and
Turkish population 13 but was comparable to other
south-eastern population like Koreans19 and Chinese
(Taiwan) 20 . Alleles IL-1RN*3 (allele 3), IL-1RN*4
(allele 4) were seen only in a few patients and
this frequency was similar to that reported in
Korean 19 and Taiwanese 20 .
Absence of any difference in allelic frequency
between patients with RA and controls has been
previously reported in studies from Europe12, 21, 22
and Taiwan 20 . We found a trend towards a lower
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frequency of homozygosity of allele 2 in patients
with RA as compared to controls even though there
was no difference in allele frequency of allele 2.
Allele 2 is associated with increased production
of IL-1Ra 23 and homozygosity may further increase
the amount of IL-1Ra produced. High IL-1Ra
downmodulates the inflammatory signals and may
thus reduce susceptibility to RA. In a study from
Korea 19 a lower frequency of allele 2 was found
in patients (2.5%) as compared to controls (7.1%).
This is in contrast to other autoimmune diseases
like SLE11, Sjögren’s syndrome12, Juvenile idiopathic
arthritis 13 where increased frequency of allele 2
is reported and it may be related to linkage with
promoters of IL-1 gene complex.
No significant difference was found in allele
frequencies, carriage rates or genotype distribution
between patients with or without extra-articular
features, seropositive or seronegative groups, early
versus delayed age of onset (< 40 yr) in our study.
Our results corroborate with an earlier study by
Cantagrel et al 21 who also did not find any
association of IL-1RA VNTR polymorphism with
disease severity. Even IL-1RN+2017T to C
polymorphism did not show any association with
disease severity 24. It is possible that once the clinical
disease sets in, the level of pro-inflammatory
cytokines is persistently higher than the
anti-inflammatory cytokines, therefore, these
polymorphisms do not make much difference 21 .
Huang et al 20 found that two individuals with allele
4 had low inflammatory activity of RA. We had
only a single patient with this allele and thus could
not study this association.
In conclusion, the IL-1RA polymorphism does
not contribute to susceptibility or presence of extraarticular features of RA in Indian patients. In this
study lower prevalence of allele 2 was found in
patients with RA and secondary Sjögren’s syndrome
and overall decreased homozygosity for allele 2
in patients with RA which needs to be confirmed
in a study with a larger sample size.
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Reprint requests: Dr A. Aggarwal, Associate Professor, Department of Immunology, Sanjay Gandhi
Postgraduate Institute of Medical Sciences, Lucknow 226014, India
e-mail: [email protected]