Downloaded from http://ard.bmj.com/ on November 25, 2014 - Published by group.bmj.
com
Ann Rheum Dis 1999;58:7–10 7
EXTENDED REPORTS
Association of oestrogen receptor gene
polymorphisms with age at onset of rheumatoid
arthritis
Toshio Ushiyama, Kanji Mori, Koji Inoue, Jie Huang, Junichi Nishioka, Sinsuke Hukuda
Abstract strual cycles.1–3 The onset of symptoms is often
Objective—In view of the possible role of associated with menopausal transition.4 With
oestrogens in the pathogenesis of rheuma- these clinical observations, the role of sex hor-
toid arthritis (RA), this study investigated mones such as oestrogens in the pathogenesis
the association between oestrogen recep- of RA has been discussed.5
tor (OR) gene polymorphisms and RA. On the other hand, a genetic factor in RA is
Methods—Pvu II and Xba I restriction also well recognised. It has been reported that
fragment length polymorphisms of the OR 15.4% of the monozygotic twins were disease
gene were analysed in 70 male and 240 concordant for RA.6 Recently, the existence of
female patients with RA, and in 300 male an oestrogen receptor (OR) gene polymor-
and 350 female controls. The absence or phism has been made clear, and its association
presence of restriction sites were repre- to some variant OR genotypes with breast
sented as P, p (Pvu II ) or X, x (Xba I ). The cancer,7 8 hypertension,9 osteoporosis,10 and
distribution of OR genotypes was com- generalised osteoarthritis11 has been reported.
pared between the RA and control sub- Assuming that individual reaction to oestro-
jects by sex. RA patients were divided into gens is genetically determined by OR gene
subgroups according to their OR geno- polymorphisms, we hypothesised that some
types, then the age at onset, seropositivity, variant of OR genotypes would be related to
and rheumatoid nodule positivity were the onset of RA in some age periods character-
compared between the subgroups. ised by hormonal status. In consideration of the
Results—The OR genotype frequency of immunomodulatory eVects of oestrogens,12 we
distribution did not have significant dif- further hypothesised that the OR genotypes
ferences between the male RA and male might be related to the severity variables such
controls nor between the female RA and as seropositivity and rheumatoid nodule posi-
female controls. In women with RA, there tivity. To ascertain these hypotheses, we inves-
tigated the relation of OR gene polymorphisms
was a significant diVerence of age at onset
with clinical variables of RA.
between the subgroups (uncorrected
p=0.047, corrected p=0.94). Female pa-
tients with the OR genotype PPxx (homo- Methods
zygote of Px) tended to have developed RA SUBJECTS
at a younger age, whereas those with A total of 310 patients with RA who visited our
PPXX and ppxx (lack of Px haplotype) outpatient clinic between October 1996 and
developed RA at an older age. In men with March 1997 were studied. They all fulfilled the
RA, there was no association between the American College of Rheumatology 1987
OR genotype and age at onset. In sero- criteria.13 Seventy were men and 240 were
positivity and rheumatoid nodule positiv- women. Two women with disease onset under
ity, there was no significant diVerence age 16 were included in this study. The clinical
Department of features of these patients such as age at onset,
Orthopaedic Surgery, between subgroups for either sex.
Shiga University of Conclusions—Some variants of the OR seropositivity, and the presence of nodules were
Medical Science, Seta, gene are related to the onset of RA in recorded. Determination of the age at onset
Otsu, 520-–2192, Japan women in certain age periods, suggesting was based on the patient’s recollection. Periph-
T Ushiyama the role of the interaction between the OR
K Mori Table 1 Characteristics of RA
K Inoue
gene and serum concentrations of oestro-
J Huang gen at the onset of the disease. Male RA Female RA
J Nishioka (Ann Rheum Dis 1999;58:7–10) (n=70) (n=240)
S Hukuda
Age (y) 59.6 (22–82) 56.6 (25–85)
Age at onset (y) 50.3 (18–82) 45.2 (11–81)*
Correspondence to: Rheumatoid arthritis (RA) is an autoimmune Seropositivity (%) 92.9 92.5
Dr T Ushiyama. Rheumatoid nodule
disease that aVects women more often than
positivity (%) 12.9 13.3
Accepted for publication men, and the severity of their symptoms is
8 October 1998 influenced by pregnancy, delivery, and men- * p = 0.007.
Downloaded from http://ard.bmj.com/ on November 25, 2014 - Published by group.bmj.com
8 Ushiyama, Mori, Inoue, et al
Table 2 Frequency of OR genotypes and relative risks for developing RA
Men Women
OR genotypes RA % (n) Control % (n) Odds ratio (95% CI) p value RA % (n) Control % (n) Odds ratio (95% CI) p value
PPXX 4.3 (3) 3.7 (11) 1.18 (0.32, 4.33) 0.81 2.9 (7) 2.9 (10) 1.02 (0.38, 2.72) 0.97
PPXx 4.3 (3) 8.3 (25) 0.49 (0.14, 1.68) 0.26 8.8 (21) 10.3 (36) 0.84 (0.48, 1.47) 0.54
PPxx 11.4 (8) 5.7 (17) 2.15 (0.89, 5.20) 0.09 6.3 (15) 4.3 (15) 1.49 (0.71, 3.11) 0.29
PpXx 15.7 (11) 23.0 (69) 0.62 (0.31, 1.25) 0.19 22.1 (53) 20.9 (73) 1.08 (0.72, 1.60) 0.72
Ppxx 37.2 (26) 26.3 (79) 1.65 (0.95, 2.86) 0.07 25.0 (60) 26.3 (92) 0.93 (0.64, 1.36) 0.73
ppxx 27.1 (19) 33.0 (99) 0.76 (0.42, 1.35) 0.34 34.9 (84) 35.3 (124) 0.98 (0.70, 1.38) 0.91
95% CI: 95% confidence intervals.
Table 3 Age at onset, seropositivity, and rheumatoid nodule positivity by OR genotypes
Male RA Female RA
RN (+) % RN (+) %
OR genotypes n Age at onset (y) RF (+) % (n) (n) n Age at onset (y) RF (+) % (n) (n)
PPXX 3 50.0 66.7 (2) 0.0 (0) 7 53.1 85.7 (6) 0.0 (0)
PPXx 3 45.0 100.0 (3) 33.3 (1) 21 42.7 100.0 (21) 4.8 (1)
PPxx 8 52.8 100.0 (8) 25.0 (2) 15 40.5 80.0 (12) 6.7 (1)
PpXx 11 49.8 100.0 (11) 18.2 (2) 53 42.3 92.5 (49) 7.5 (4)
Ppxx 26 51.7 88.5 (23) 11.5 (3) 60 45.6 91.7 (55) 20.0 (12)
ppxx 19 48.5 94.7 (18) 5.3 (1) 84 47.6 94.0 (79) 16.7 (14)
p value 0.94 0.19 0.33 0.047 0.98 0.36
RF: rheumatoid factor, RN: rheumatoid nodule.
eral blood was collected for the analysis of OR The diVerence in the continuous variable
gene polymorphisms. The blood samples of between two groups was evaluated by a
300 men (aged 17–91, mean 48.9) and 350 Student t test, while the diVerences between
women (20–87, mean 43.5) who underwent an more than three groups was evaluated by
annual health care check held by several com- analysis of variance (ANOVA). A ÷2 test was
munities were used as a control. also used when appropriate. The software used
for the analysis was SPSS (SPSS Inc, Chicago,
OR GENE POLYMORPHISM ANALYSIS USA).
An analysis of OR gene restriction fragment
length polymorphisms (RFLPs) in the intron 1 Results
was performed according to the methods of the CHARACTERISTICS OF RA PATIENTS
previous report.10 Briefly, 100 ng of genomic Table 1 summarises by sex the clinical charac-
DNA, extracted from peripheral blood, was teristics of the RA patients in the study. Their
amplified by a polymerase chain reaction ages ranged from 22 to 82 (mean 59.6) for men
(PCR) using 2.5 units of Ex Taq polymerase and from 25 to 85 (mean 56.6) for women. The
with the accompanying buVer, 2.5 mM each of age at onset of RA was from 18 to 82 (mean
dNTP (TAKARA SHUZO, Otsu, Japan ) and 50.3) for men and from 11 to 81 (mean 45.2)
0.2 µM each of specific oligonucleotide prim- for women, the diVerence between both sexes
ers in the same PCR conditions.10 The product being significant (p=0.007). Serum rheuma-
contains a part of intron 1 and exon 2 of the toid factor positivity was 92.9% for men and
OR gene. The amplified PCR products were 92.5% for women, while rheumatoid nodule
digested with a restriction endonuclease, Pvu II positivity was 12.9% for men and 13.3% for
or Xba I (TAKARA SHUZO) and electro- women. Frequencies of OR genotypes in over-
phoresed on 1.0% agarose gel. The RFLPs all RA RFLPs of the OR gene were classified
were represented by P and p (Pvu II ), and X into six genotypes according to the combina-
and x (Xba I ), with capital and small letters tion of both digestion patterns of Pvu II and
signifying the absence or presence of restriction Xba I: PPXX, PPXx, PPxx, PpXx, Ppxx and
sites, respectively. ppxx. We could not find the three genotypes,
PpXX, ppXX and ppXx in any of the subjects.
STATISTICAL ANALYSIS The frequencies of OR genotypes in RA
The frequency of distribution of each genotype patients and controls are shown by sex in table
was compared between the RA group and con- 2. The frequencies in both the male and female
trols by sex. An odds ratio and 95% confidence controls were essentially the same as those pre-
intervals (CI) were calculated with respect to viously reported for Japanese women.10 There
the presence of the reference OR genotype. was no significant diVerence between the male
Table 4 Age at onset, seropositivity, and rheumatoid nodule positivity by Px haplotypes
Male RA (n=59) Female RA (n=187)
Px haplotypes n Age at onset RF (+) % (n) RN (+) % (n) n Age at onset RF (+) % (n) RN (+) % (n)
11 8 52.8 100.0 (8) 25.0 (2) 15 40.5 80.0 (12) 6.7 (1)
10 29 51.0 89.7 (26) 13.8 (4) 81 44.8 93.8 (76) 16.0 (13)
00 22 48.7 90.9 (20) 4.5 (1) 91 48.0 93.4 (85) 15.4 (14)
p value 0.75 0.15 0.13 0.056 0.88 0.69
Px haplotype 11 (Px/Px): Ppxx; 10 (Px/-): PPXx, Ppxx; 00 (-/-): PPXX, ppxx.
Downloaded from http://ard.bmj.com/ on November 25, 2014 - Published by group.bmj.com
Oestrogen receptor gene polymorphisms and RA 9
40
Px haplotype
11
10
30 00
Percentage
20
10
0
–19 20–29 30–39 40–49 50–59 60–69 70–
Age at onset
Figure 1 Distribution of age at onset of RA in women by Px haplotypes.
RA patients and male controls nor between the Figure 1 shows the distribution of age at onset
female RA patients and female controls in the for these three groups. The peak age at onset in
frequency of any genotype. patients having haplotype 11 is seen at the
fourth and fifth decade, in those having 10 at
CLINICAL VARIABLES OF RA IN EACH OR the fifth decade, and in those having 00 at the
GENOTYPE fifth and sixth decade.
Patients with RA were divided into six groups
according to OR genotypes by sex. Age at Discussion
onset, seropositivity, and rheumatoid nodule This study suggests that some of the OR gene
positivity were compared between the groups polymorphisms were related to the age at onset
(table 3). There was no significant diVerence in women with RA. There was a significant dif-
between the groups in male RA age at onset, ference between six genotype groups in female
however there was in the female RA groups RA age at onset. Female patients with Px hap-
(uncorrected p=0.047 using ANOVA, cor- lotype 11 tended to have developed the disease
rected p=0.94). The mean age at onset for the at a younger age, those with Px haplotype 10 at
patients with OR genotype PPxx was 40.5, an intermediate age and those with Px
PpXx 42.3, PPXx 42.7, Ppxx 45.6, ppxx 47.6, haplotype 00 at a relatively older age, suggest-
and PPXX 53.1, respectively. Post hoc test by ing that the Px allelic haplotype may play some
ScheVe’s method, however, showed no signifi- part in the onset of the disease in women.
cant diVerence of mean age at onset between The serum oestrogen concentration in
groups with a significant level of 0.05. Regard- women is highest during childbearing age, and
ing seropositivity and rheumatoid nodule posi- then rapidly decreases after menopause. Al-
tivity, there was no significant diVerence found though we did not examine menopausal status
between groups of either sex by the ÷2 test. at disease onset, the peak age at onset of female
In a separate analysis, patients with RA were RA, having Px haplotype 11, was about the
grouped into three groups according to the Px premenopausal period, whereas that of women
allelic haplotype, in which the genotype PPxx is having Px haplotype 00 about the perimeno-
a homozygote (Px haplotype 11), PPXx and pausal or postmenopausal period. Given that
Ppxx are heterozygotes (Px haplotype 10), and variants of OR gene polymorphisms would
PPXX and ppxx would be characterised by the determine the reaction to hormonal stimuli,
lack of the Px haplotype (Px haplotype 00).10 the coexistence of such a genetic predisposition
Because the haplotype of PpXx is unclear, and exposure to a specific hormonal environ-
patients with this genotype were excluded from ment might be related to the onset of RA.
this analysis. Age at onset, seropositivity, and The characteristic clinical features of older
rheumatoid nodule positivity were then com- age onset RA have been described by many
pared between these groups (table 4). There authors.14 15 An equal distribution of the
was no significant diVerence in any of these disease between men and women, a tendency
clinical parameters for both sexes. However, in towards large joint involvement, an increased
female RA, three groups tended to have a erythrocyte sedimentation rate, and less sero-
diVerent age at onset (uncorrected p=0.056 positivity have been mentioned as characteris-
using ANOVA, corrected p=0.11). The mean tic manifestations of older age onset RA. This
ages at disease onset in Px haplotype 11, 10, study provides a diVerent genetic susceptibility
and 00, were 40.5, 44.8, and 48.0, respectively. to the development of RA between younger
Downloaded from http://ard.bmj.com/ on November 25, 2014 - Published by group.bmj.com
10 Ushiyama, Mori, Inoue, et al
and older onset female RA, suggesting a diVer- bias. In addition, if p values are corrected for
ent pathogenesis is involved in the two multiple comparison, they are no longer
subgroups. significant. Therefore, the results of this study
It is noteworthy that Px haplotype 11 was should not be considered as proof of an
found to be a risk factor for osteoporosis, association, but as evidence for a putative asso-
whereas Px haplotype 00 had a preventive role ciation that needs to be confirmed in a
in a previous study.10 Osteoporosis commonly separate, larger study.
seen in RA has been thought to be caused by
disuse, use of corticosteroids, and rheumatoid We thank Dr M Nabae, Dr T Mori, Dr T Takase, Dr T Kawa-
inflammation.16 Our study suggests that the saki, Dr G Yoshikawa, Dr T Imanaka, Mrs K Taniguchi, and
Mrs K Nishikawa for collecting blood samples.
genetic predisposition for RA is also related to Funding: this study was supported in part by grants-in-aid for
osteoporosis in some subgroups of RA. scientific research (07807139) from the Ministry of Education,
Science, and Culture of Japan.
Loss of ovarian function causes a precipitous
loss of bone that can be prevented by oestrogen 1 Lawrence JS. Rheumatism in populations. London: Hein-
replacement. Although cellular and biochemi- emann, 1977.
2 Persellin RH. The eVect of pregnancy on rheumatoid
cal changes mediate the adverse eVects of oes- arthritis. Bull Rheum Dis 1976;27:922–7.
trogen deficiency, it has been shown that 3 Rudge SR, Kowanko IC, Drury PL. Menstrual cyclicity of
finger joint size and grip strength in patients with rheuma-
oestrogen loss up-regulates osteoclastogenesis toid arthritis. Ann Rheum Dis 1983;42:425–30.
through an increase in the production of inter- 4 Goemaere S, Ackerman C, Goethals K, De Keyser F, Van
der Straeten C, Verbruggen G, et al. Onset of symptoms of
leukin 6 (IL6) in the microenvironment of the rheumatoid arthritis in relation to age, sex and menopausal
bone tissue, resulting in osteoporosis.17 On the transition. J Rheumatol 1990;17:1620–2.
5 Masi AT. Sex hormones and rheumatoid arthritis: cause or
other hand, the concentration of IL6 is eVect relationships in a complex pathophysiology? Clin
increased in the synovial fluids of RA patients, Exp Rheumatol 1995;13:227–40.
6 Silman AJ, MacGregor AJ, Thomson W, Holligan S, Carthy
and IL6 is believed to play important parts in D, Farhan A, et al. Twin concordance rates for rheumatoid
immune response and activation of inflamma- arthritis: results from a nationwide study. Br J Rheumatol
1993;32:903–7.
tion at articular joints.18 It is already known that 7 Hill SM, Fuqua SAW, Chamness GC, Greene GL, McGuire
articular chondrocytes and synovial cells ex- WL. Estrogen receptor expression in human breast cancer
associated with an estrogen receptor gene restriction
press OR19–22 and that oestrogen induces the fragment length polymorphism. Cancer Res 1989;49:
up-regulation of IL6 production by chondro- 145–8.
8 Yaich L, Dupont WD, Cavener DR, Parl FF. Analysis of the
cytes, showing a conspicuous diVerence from Pvu II restriction fragment-length polymorphism and exon
bone tissue.23 It is therefore probable that structure of the estrogen receptor gene in breast cancer and
peripheral blood. Cancer Res 1992;52:77–83.
women who show a specific reaction to the 9 Lehrer S, Rabin J, Kalir T, Schachter BS. Estrogen receptor
hormonal stimuli may tend to develop either variant and hypertension in women. Hypertension 1993;
21:439–41.
osteoporosis in bone tissue at the postmeno- 10 Kobayashi S, Inoue S, Hosoi T, Ouchi Y, Shiraki M, Orimo
pausal period or rheumatoid inflammation in H. Association of bone mineral density with polymorphism
of the estrogen receptor gene. J Bone Miner Res 1996;11:
joint tissues at the premenopausal period 306–11.
through diVerent eVects of oestrogen toward 11 Ushiyama T, Ueyama H, Inoue K, Nishioka J, Ohkubo I,
Hukuda S. Estrogen receptor gene polymorphism and gen-
IL6 production. eralized osteoarthritis. J Rheumatol 1998;25:134–7.
Interestingly, the age at onset of RA has been 12 Cutolo M, Sulli A, Seriolo B, Accardo S, Masi AT.
Estrogens, the immune response and autoimmunity. Clin
increasing in Japan over a 30 year period.24 Exp Rheumatol 1995;13:217–26.
Previously, women of childbearing age were 13 Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries
JF, Cooper NS, et al. The American Rheumatism Associa-
most often aVected, but now the peak has tion 1987 revised criteria for the classification of rheuma-
shifted to the perimenopausal age. This toid arthritis. Arthritis Rheum 1988;31:315–24.
14 Inoue K, Shichikawa K, Nishioka J, Hirota S. Older age
increase in age at onset of RA in Japan is prob- onset rheumatoid arthritis with or without osteoathritis.
ably reducible to some environmental changes Ann Rheum Dis 1987;46:908–11.
15 Terkeltaub R, Esdaile J, Décary F, Tannenbaum H. A clini-
that have occurred during these past years. cal study of older age onset rheumatoid arthritis with com-
Given that age at onset of RA is influenced by parison to an early onset group. J Rheumatol 1983;10:418–
24.
hormonal status, this study suggests that a 16 Deodhar AA, Woolf AD. Bone mass measurement and bone
change in hormonal environment, such as a metabolism in rheumatoid arthritis: a review. Br J Rheuma-
tol 1996;35:309–22.
decrease in childbearing, may explain this phe- 17 Jilka RL, Hangoc G, Girasole G, Passeri G, Williams DC,
nomenon. Abrams JS, et al. Increased osteoclast development after
estrogen loss: mediation by interleukin-6. Science 1992;
No significant relation between OR geno- 257:88–91.
types and disease severity indices was found in 18 Ito A, Itoh Y, Sasaguri Y, Morimatsu M, Mori Y. EVects of
interleukin-6 on the metabolism of connective tissue com-
this study. It is to be noted that the number of ponents in rheumatoid synovial fibroblasts. Arthritis
seronegative patients and of those with nodules Rheum 1992;35:1197–201.
19 Young PCM, Stack MT. Estrogen and glucocorticoid recep-
were too small to draw a negative conclusion. tors in adult canine articular cartilage. Arthritis Rheum
Specific OR genotypes may directly aVect 1982;25:568–73.
20 Rosner IA, Manni A, Malemud CJ, Boja B, Moskowitz RW.
the levels of expression through transcriptional Estradiol receptors in articular chondrocytes. Biochem
regulation, resulting in disease onset, or it is Biophys Res Commun 1982;106:1378–82.
21 Cutolo M, Accardo S, Villaggio B, Clerico P, Bagnasco M,
possible that they may be indirectly related to Coviello DA, et al. Presence of estrogen-binding sites on
another responsible gene, such as HLA gene, macrophage-like synoviocytes and CD8+, CD29+,
CD45RO+ T lymphocytes in normal and rheumatoid syn-
because both the OR and HLA genes are ovium. Arthritis Rheum 1993;36:1087–97.
mapped in chromosome 6. The exact mecha- 22 Ushiyama T, Inoue K, Nishioka J. Expression of estrogen
receptor related protein (p29) and estradiol binding in
nism of the relation between OR gene polymor- human arthritic synovium. J Rheumatol 1995;22:421–6.
phisms and the age at onset of RA therefore 23 Guerne PA, Carson DA, Lotz M. IL-6 production by
human articular chondrocytes. J Immunol 1990;144:499–
needs to be further investigated. 505.
In this study, determination of the age at 24 Imanaka T, Shichikawa K, Inoue K, Shimaoka Y, Takenaka
onset was based on the patient’s recollection, Y, Wakitani S. Increase in age at onset of rheumatoid
arthritis in Japan over a 30 year period. Ann Rheum Dis
probably resulting in inclusion of some recall 1997;56:313–16.
Downloaded from http://ard.bmj.com/ on November 25, 2014 - Published by group.bmj.com
Association of oestrogen receptor gene
polymorphisms with age at onset of rheumatoid
arthritis
Toshio Ushiyama, Kanji Mori, Koji Inoue, Jie Huang, Junichi Nishioka and
Sinsuke Hukuda
Ann Rheum Dis 1999 58: 7-10
doi: 10.1136/ard.58.1.7
Updated information and services can be found at:
http://ard.bmj.com/content/58/1/7
These include:
References This article cites 23 articles, 10 of which you can access for free at:
http://ard.bmj.com/content/58/1/7#BIBL
Email alerting Receive free email alerts when new articles cite this article. Sign up in the
service box at the top right corner of the online article.
Topic Articles on similar topics can be found in the following collections
Collections Connective tissue disease (3673)
Musculoskeletal syndromes (4279)
Rheumatoid arthritis (2804)
Degenerative joint disease (4004)
Immunology (including allergy) (4368)
Genetics (844)
Notes
To request permissions go to:
http://group.bmj.com/group/rights-licensing/permissions
To order reprints go to:
http://journals.bmj.com/cgi/reprintform
To subscribe to BMJ go to:
http://group.bmj.com/subscribe/
