- Email: [email protected]
The TNF2 allele does not contribute towards susceptibility to systemic lupus erythematosus
Immunology
Letters
55 (1997)
I ~3
The TNF2 allele does not contribute towards susceptibility to systemic lupus erythematosus
Received
30 August
1996: revised
7 October
1996
Abstract The uncommon allele (TNFZ) of a polymorphism in the promoter region of the tumor necrosis factor-alpha (TNF-x) gene has been reported to be increased in Caucasian systemic lupus erythematosus (SLE) patients (associated with HLA-DR3). To investigate whether TNF? contributes towards susceptibility to Chinese SLE patients (not associated with HL,4-DR3). 100 patients with SLE and 107 controls were studied. The frequency of TNF3 allele in controls was 0.140. There was a strong association between TNF2 allele and HLA-DR3 (P < 10 “) in controls. The frequency of TNFZ allele in SLE patients was 0.15. There was no difference in frequencies of TNF2 allele between patients and controls. This finding strongly suggests that TNF? does not play a direct role in the susceptibility of SLE. C, 1997 Elsevier Science B.V. KPJWW~S: TNFZ:
Systemic
lupus erythematosus;
Tumor
necrosis
1. Introduction Systemic lupus erythematosus (SLE) is a severe autoimmune disease with genetic susceptibility [l-3]. Most studies implicate that genes within the major histocompatibility complex (MHC) may play an important role. Many reports have indicated HLA B8-DR3 or B7-DRZ to be strongly associated with SLE [4,5]. However, apart from those of the HLA class 1 and II genes, whether other alleles contribute to the susceptibility of SLE is an important issue. Tumor necrosis factor-alpha (TNF-a) is a central mediator of inflammatory and immunological response, and so has been speculated to contribute to the susceptibility of MHC-related autoimmune diseases [6]. In * Corresponding + 886 7 31 18111.
author.
Tel.:
+ 886 7 3121101 (ext. 6064): fax:
Ol65-2478:97 $17.1)0 i( 1997 Elsevier Science P/f S0l6~-~-17X~96)0263~-1
B.V. All rights
reserved.
factor
1986. the first known polymorphism within the human TNF-x gene was reported. It lies within the promoter region and involves the substitution of guanine by adenosine in the uncommon (TNFZ) allele [7]. In 1994, Wilson et al. firstly reported that the frequency of TNF:! allele increased in Caucasian SLE patients compared with that in controls, as did HLADR? [S]. But they could not demonstrate the association of HLA-DR3 or TNF3 with SLE independently of each other. Therefore, whether the TNF2 allele plays a direct role in the susceptibility of SLE remail-s unclear. In order to resolve this debate. cxaminatic, I of the TNF-x genotype in ethnic groups that demonstrate different MHC association with SLE may help to answer the question. If the TNF2 allele is associated with SLE in those individuals who do not associate with HLA-DR3, then direct involvement of TNF2 allele in the susceptibility of SLE will be strengthened.
Previous studies from East Asia have shown that SLE is not associated with HLA-DR3 in Korean [9], Japanese [lo, 111, and Chinese [ 12,131. Therefore, the study of the TNF-a genotype in East Asia in connection with SLE may be very helpful to elucidate the role of TNF2 in the susceptibility of SLE. Therefore, the frequency of TNF2 allele in Chinese SLE patients in Taiwan was investigated.
2. Materials
-107 bp - 87 bp
and methods
2 1. Patients and controls One hundred patients with SLE were enrolled in this study. All patients fulfilled the ARA 1982 revised criteria for the classification of SLE [14]. There were 90 females and 10 males. One hundred and seven unrelated healthy subjects served as controls. 2.2. Extraction
of genomic
DNA
Genomic mononuclear
DNA was extracted from peripheral blood cells using the standard method [15].
2.3. TNF-a
genotyping
TNFl and TNF2 allele typing was performed as previously described by Wilson et al. [16]. Briefly, a 107 bp stretch of the TNF-c( promoter was amplified by PCR using two primers (sense: 5’-AGGCAATAGGTTTTGAGGGCCAT-3’; antisense: 5’-TCCTCCCTGCTCCGATTCCG-3’) [ 161. The PCR product was then digested with NcoI for 24 h at 37°C and was analysed on a 4% MetaphorTM agarose (FMC BioProducts, Rockland, ME). Homozygote TNFl showed one fragment of 87 bp. Homozygote TNF2 showed a single 107 bp fragment. Heterozygote TNFl/TNF2 showed fragments of 87 and 107 bp. 2.4. HLA-DR
genotlping
HLA-DR genotyping was performed by PCR followed by Southern hybridization with a sequence specific oligonucleotide probe as described previously [ 171. 2.5. Stutisticul ardysis The 1’ test and Fisher’s exact test were used evaluate significant differences between groups.
two individuals (2%) were homozygote TNF2 (Fig. 1). The allele frequency of TNF2 was 0.140 in controls. HLA-DR genotyping was performed in 95 of 107 controls. The TNF2 allele was found to be strongly associated with HLA-DR3 positivity (P < lOOx) (Table 1). TNFl was significantly associated with HLA-DR4 and DR8. In 100 patients with SLE. 81 (81%) were homozygote TNFl , 19 ( 19’/,) were heterozygote TNF 1: TNF2 and none was homozygote TNF2. The allele frequency of TNF2 was 0.095 in patients with SLE. There was no significant difference in frequencies of TNF2 allele between patients and controls (Table 2).
4. Discussion In 168 Caucasian controls, Wilson et al. found that the frequency of the TNF2 allele was 0.17 [8]. In 107 Chinese controls, we found that the frequency of the TNF2 allele was 0.14. There was no difference in the frequency of the TNF2 allele between Caucasian and Chinese. In addition, there was a very strong association between the TNF2 allele and HLA-DR3 in Chinese, as in the Caucasians [18]. Wilson et al. reported that the frequency of the TNF2 allele increased in Caucasian SLE patients compared with controls [8]. However, in the present study, we could not find increased frequency of the TNF2 allele in our SLE pa-
to
3. Results Among 107 controls, 79 (74%) TNFl, 26 (24%) were heterozygote
Fig. I, Genotyping of TNF I and TNF2 alleles. Lane I. a standard marker product of Xl74 Hi&I: lane 2. a PCR product (107 bp stretch of the TNF-2 promotor): lane 3. the same PCR product digested with VUJI (a heterozygote TNFI;TNF2): lane 4, a homozygote TNF2 digested with Nc01; lane 5. a homozypote TNFI digested with :VcoI.
Table I Association ~..___
of HLA-DR3
with TNF?
in controls
TNF2-
TNF2+
P
I 69
1:
-._____
were homozygote TNFl/TNFZ and
DR3+ DR3-
(n = 951 . .._______~
7
s
Table
7
[i]
The frequency
of TNF3
C on~rols
in controls ___
(II = 107)
and SLE
patients
Deapen. B..
ArthI-itih SLE
(II = 100)
P
0 14(l ~______
,’ No signiticant
0.095
NS.’
T.R..
144
tients. As previously reported, Chinese SLE patients were not associated with HLA-DR3 [I?, 131. However, in Wilson’s study, their Caucasian SLE patients were strongly associated with HLA-DR3 [8]. If the TNF? allele is directly involved in the susceptibility of SLE, the increased frequency of TNF2 should appear not only in Caucasian patients (associated with HLA-DR3 I but also in Chinese patients (not associated with HLADR3). However, the fact that the increased frequency of the TNFZ allele only appears in Caucasian patients. but not in Chinese patients strongly suggests that TNF2 does not play a direct role in the susceptibility of SLE. Recently, Pociot et al. reported a new promoter polymorphism of TNF-s( gene, a G/A polymorphic sequence at position-238 [19]. Turner et al. found the microsatellite allele TNFd3 (in 3’ repressor region) was significantly associated with the capacity of the leukocyte to produce TNF-x in vitro [30]. Whether these new polymorphisms are associated with the susceptibility of SLE needs further investigation.
J.R.
[7] Yedospaao\. V.A.
V.N..
Biol.
Gordon. Emery.
IYI
2-I.
Ilong.
GH..
Kameda.
II3 apen.
H.W.
IlYU.3)
J.R..
Kaohsiung
J. Rheumatol.
14, 867-869.
Tsai. J.J.. Tsai.
W.J.. Jen. J.H.
J. Med.
Sci. 9. 664-667.
!_.I ._
L.I\.
‘llld
IHarhcll- S\mp
C.k~n(.
F- S.. dc Vw\.
Du11‘. (i W
N.. \ an dc
Il’)‘JJI
I ur
J
ln-
Takcucht.
t-.. U;lkancl.
K..
k ,~mada.
K.. lt~~. K and Park. K.?.
447.
H.. Ma~aumolo.
tl..
Taha\ah~.
P.I. and I,*;I~I.
7 ., Uaw.
\i’. (IYSFI
Kaji>;lma.
Kunihir~~.
S.. Tcraahf. l‘anaha.
K..
S. ,~nd \‘,~nc\c.
h..
)
T. (1’8
Ti$\uc
.Anti-
Lan and
Wang.
K..
Wony.
Serjeantwn.
.
R.W.S
I l‘jS71J.
S.W
Ilo
C‘han.
Rheumatol
K IA.
Ireland. K.I.
Cohen. N.F.,
,\.CJ..
M ang.
I’..
c1W2) ,\thrllls
I).
Wilson.
D)he\.
\‘ccr-
Rhcum
35. 17’1 D.D.
hla\i.
Talal.
antI
3.
s\.<;..
di Ciovinc.
(lYY2)
\en.
J.H..
(‘hw.
J
Rheumatol.
Llum.
Mol. J.R.,
I‘S,.
I’~~lc&>
D’Alfonso. P.M.
D.M..
t;ktion
60.
11.1’
Ralhcm~~rc.
Grnct.
D.J R.J.
~l’)Shl
Nucluc
Tsa.
(1995) Grant.
Sinnoll.
C.W S.. Stand. S.C.D..
W.J..
-Twt.
J.J. ;IICI
l.lu.
If.%.
14%.
( 1903)
do (ii
Cornpaw). J
S..
Immunul.
I.amb.
P.J. and I Iutchinwl.
I I I3 I I 17.
.tnd hl’l.
-\.l.t
I. 35.:.
22. IJIO
nnd Dulf:
t‘..
P.A..
Mcsl~anc. Winchcstcr.
17::.
h\.G.. de Vi-ies. N.. PocI~~. I. L.B.A.
Dyer.
/CT.. and
16. 1715.
G.W.
Wilson.
J.l..
J.G..
Rheum.
S.A.. Rc\.
E.S.. Fric\.
Schaller.
Arthritis
T‘urncr.
R.. Dcmanlnc. and \‘crgar\i.
h46
E.hl..
,\cid\
WI and Lio.
Dt>-
Bold)rc\a.
.Shingaro\a.
SprlnE.
Marc.
V.(;..
22.
D.G..
Richiardi. (‘hen
,\.M..
21, 342
S.N..
K.. Welsh.
(1981)
M.C’. (1987)
N.S..
H.. Tokunag.
S.. Jimi.
H
[I31 Doherty.
1191 Pociol.
1I.M..
ICot-obukt,.
Ilil.
Putre.
Hochberg.
R.L..
;5.
12. 9 19 Y2i.
B.R..
Dunchle!.
(IYY5)
[2] yeh.
Turc\r\ha!a.
By>tro\.
P. and
\r’.. Hirose.
!g?ns 31. 221
[I51 Miller.
[I]
D.;4..
.,4ntiyn\
c‘.. di C;io\inc.
H.\r’..
S.. :\ito.
Ha\shin\.
S.,I..
K.. Han.
Nishi~ouri.
Rothfield.
References
Iwnberg.
191. Kim.
Mat\uda.
K..
[I81
C’. :2..
(;.I’..
( 19X6)(‘old
L.B.A..
I. Rheumatol.
[I71
R1. and
255.
I 624.
\‘.. Shioha~~a.
[IhI
(I‘~‘)‘)
c1940)Tiysuc
A.h..
Chumakov.
f 199.4) J. Rheumatol. [I’)1 tlashim~,to. I-I.. Twda.
Acknowledgements
h1.J
Georgia.
.\.G..
111tl1101.
[I4
T.M.
K.. Qu~nlan.
6. 247 Warner.
Shakho\.
\1’..4.
ho. 61
Wilson. Putts.
[I II
Bolahrishnan.
Marker?
Filippov.
S.A..
35. 6JI
Lin t’or excellent technical assisFang for preparation of this
L.S..
M.M..
Ovchinniko\.
tl..
I>.. Bachman.
Ma&..
318.
Walport,
S.4..
C‘hu\pilo.
PI
and
A
A.H.L..
and
.Ari7o\.
brynin.
PI
I_.. HotwIt/.
Walker.
137.
[131 Tan.
We thank (‘hia-Hui tance and Ai-Hwei manuscript.
Beischet. Fielder.
Bat&lot-.
A., Wcinrib.
35, il I
C’.D. (19x81 Dis.
[_‘I So. .A.K.L..
dilf‘erencc.
P..
Rheum.
[J] Welch. West.
TNF
D.. Escalante.
Rol-Burman.
1 S . Vati dct 1'7. 557
Scar/a.
47. 501
W.R.. I.\’
R.
,~nd
511-1.
t3rcnchIc~. (l+)S)
Ml.
I’ L.C’..
Transplaw
Letters
55 (1997)
I ~3
The TNF2 allele does not contribute towards susceptibility to systemic lupus erythematosus
Received
30 August
1996: revised
7 October
1996
Abstract The uncommon allele (TNFZ) of a polymorphism in the promoter region of the tumor necrosis factor-alpha (TNF-x) gene has been reported to be increased in Caucasian systemic lupus erythematosus (SLE) patients (associated with HLA-DR3). To investigate whether TNF? contributes towards susceptibility to Chinese SLE patients (not associated with HL,4-DR3). 100 patients with SLE and 107 controls were studied. The frequency of TNF3 allele in controls was 0.140. There was a strong association between TNF2 allele and HLA-DR3 (P < 10 “) in controls. The frequency of TNFZ allele in SLE patients was 0.15. There was no difference in frequencies of TNF2 allele between patients and controls. This finding strongly suggests that TNF? does not play a direct role in the susceptibility of SLE. C, 1997 Elsevier Science B.V. KPJWW~S: TNFZ:
Systemic
lupus erythematosus;
Tumor
necrosis
1. Introduction Systemic lupus erythematosus (SLE) is a severe autoimmune disease with genetic susceptibility [l-3]. Most studies implicate that genes within the major histocompatibility complex (MHC) may play an important role. Many reports have indicated HLA B8-DR3 or B7-DRZ to be strongly associated with SLE [4,5]. However, apart from those of the HLA class 1 and II genes, whether other alleles contribute to the susceptibility of SLE is an important issue. Tumor necrosis factor-alpha (TNF-a) is a central mediator of inflammatory and immunological response, and so has been speculated to contribute to the susceptibility of MHC-related autoimmune diseases [6]. In * Corresponding + 886 7 31 18111.
author.
Tel.:
+ 886 7 3121101 (ext. 6064): fax:
Ol65-2478:97 $17.1)0 i( 1997 Elsevier Science P/f S0l6~-~-17X~96)0263~-1
B.V. All rights
reserved.
factor
1986. the first known polymorphism within the human TNF-x gene was reported. It lies within the promoter region and involves the substitution of guanine by adenosine in the uncommon (TNFZ) allele [7]. In 1994, Wilson et al. firstly reported that the frequency of TNF:! allele increased in Caucasian SLE patients compared with that in controls, as did HLADR? [S]. But they could not demonstrate the association of HLA-DR3 or TNF3 with SLE independently of each other. Therefore, whether the TNF2 allele plays a direct role in the susceptibility of SLE remail-s unclear. In order to resolve this debate. cxaminatic, I of the TNF-x genotype in ethnic groups that demonstrate different MHC association with SLE may help to answer the question. If the TNF2 allele is associated with SLE in those individuals who do not associate with HLA-DR3, then direct involvement of TNF2 allele in the susceptibility of SLE will be strengthened.
Previous studies from East Asia have shown that SLE is not associated with HLA-DR3 in Korean [9], Japanese [lo, 111, and Chinese [ 12,131. Therefore, the study of the TNF-a genotype in East Asia in connection with SLE may be very helpful to elucidate the role of TNF2 in the susceptibility of SLE. Therefore, the frequency of TNF2 allele in Chinese SLE patients in Taiwan was investigated.
2. Materials
-107 bp - 87 bp
and methods
2 1. Patients and controls One hundred patients with SLE were enrolled in this study. All patients fulfilled the ARA 1982 revised criteria for the classification of SLE [14]. There were 90 females and 10 males. One hundred and seven unrelated healthy subjects served as controls. 2.2. Extraction
of genomic
DNA
Genomic mononuclear
DNA was extracted from peripheral blood cells using the standard method [15].
2.3. TNF-a
genotyping
TNFl and TNF2 allele typing was performed as previously described by Wilson et al. [16]. Briefly, a 107 bp stretch of the TNF-c( promoter was amplified by PCR using two primers (sense: 5’-AGGCAATAGGTTTTGAGGGCCAT-3’; antisense: 5’-TCCTCCCTGCTCCGATTCCG-3’) [ 161. The PCR product was then digested with NcoI for 24 h at 37°C and was analysed on a 4% MetaphorTM agarose (FMC BioProducts, Rockland, ME). Homozygote TNFl showed one fragment of 87 bp. Homozygote TNF2 showed a single 107 bp fragment. Heterozygote TNFl/TNF2 showed fragments of 87 and 107 bp. 2.4. HLA-DR
genotlping
HLA-DR genotyping was performed by PCR followed by Southern hybridization with a sequence specific oligonucleotide probe as described previously [ 171. 2.5. Stutisticul ardysis The 1’ test and Fisher’s exact test were used evaluate significant differences between groups.
two individuals (2%) were homozygote TNF2 (Fig. 1). The allele frequency of TNF2 was 0.140 in controls. HLA-DR genotyping was performed in 95 of 107 controls. The TNF2 allele was found to be strongly associated with HLA-DR3 positivity (P < lOOx) (Table 1). TNFl was significantly associated with HLA-DR4 and DR8. In 100 patients with SLE. 81 (81%) were homozygote TNFl , 19 ( 19’/,) were heterozygote TNF 1: TNF2 and none was homozygote TNF2. The allele frequency of TNF2 was 0.095 in patients with SLE. There was no significant difference in frequencies of TNF2 allele between patients and controls (Table 2).
4. Discussion In 168 Caucasian controls, Wilson et al. found that the frequency of the TNF2 allele was 0.17 [8]. In 107 Chinese controls, we found that the frequency of the TNF2 allele was 0.14. There was no difference in the frequency of the TNF2 allele between Caucasian and Chinese. In addition, there was a very strong association between the TNF2 allele and HLA-DR3 in Chinese, as in the Caucasians [18]. Wilson et al. reported that the frequency of the TNF2 allele increased in Caucasian SLE patients compared with controls [8]. However, in the present study, we could not find increased frequency of the TNF2 allele in our SLE pa-
to
3. Results Among 107 controls, 79 (74%) TNFl, 26 (24%) were heterozygote
Fig. I, Genotyping of TNF I and TNF2 alleles. Lane I. a standard marker product of Xl74 Hi&I: lane 2. a PCR product (107 bp stretch of the TNF-2 promotor): lane 3. the same PCR product digested with VUJI (a heterozygote TNFI;TNF2): lane 4, a homozygote TNF2 digested with Nc01; lane 5. a homozypote TNFI digested with :VcoI.
Table I Association ~..___
of HLA-DR3
with TNF?
in controls
TNF2-
TNF2+
P
I 69
1:
-._____
were homozygote TNFl/TNFZ and
DR3+ DR3-
(n = 951 . .._______~
7
s
Table
7
[i]
The frequency
of TNF3
C on~rols
in controls ___
(II = 107)
and SLE
patients
Deapen. B..
ArthI-itih SLE
(II = 100)
P
0 14(l ~______
,’ No signiticant
0.095
NS.’
T.R..
144
tients. As previously reported, Chinese SLE patients were not associated with HLA-DR3 [I?, 131. However, in Wilson’s study, their Caucasian SLE patients were strongly associated with HLA-DR3 [8]. If the TNF? allele is directly involved in the susceptibility of SLE, the increased frequency of TNF2 should appear not only in Caucasian patients (associated with HLA-DR3 I but also in Chinese patients (not associated with HLADR3). However, the fact that the increased frequency of the TNFZ allele only appears in Caucasian patients. but not in Chinese patients strongly suggests that TNF2 does not play a direct role in the susceptibility of SLE. Recently, Pociot et al. reported a new promoter polymorphism of TNF-s( gene, a G/A polymorphic sequence at position-238 [19]. Turner et al. found the microsatellite allele TNFd3 (in 3’ repressor region) was significantly associated with the capacity of the leukocyte to produce TNF-x in vitro [30]. Whether these new polymorphisms are associated with the susceptibility of SLE needs further investigation.
J.R.
[7] Yedospaao\. V.A.
V.N..
Biol.
Gordon. Emery.
IYI
2-I.
Ilong.
GH..
Kameda.
II3 apen.
H.W.
IlYU.3)
J.R..
Kaohsiung
J. Rheumatol.
14, 867-869.
Tsai. J.J.. Tsai.
W.J.. Jen. J.H.
J. Med.
Sci. 9. 664-667.
!_.I ._
L.I\.
‘llld
IHarhcll- S\mp
C.k~n(.
F- S.. dc Vw\.
Du11‘. (i W
N.. \ an dc
Il’)‘JJI
I ur
J
ln-
Takcucht.
t-.. U;lkancl.
K..
k ,~mada.
K.. lt~~. K and Park. K.?.
447.
H.. Ma~aumolo.
tl..
Taha\ah~.
P.I. and I,*;I~I.
7 ., Uaw.
\i’. (IYSFI
Kaji>;lma.
Kunihir~~.
S.. Tcraahf. l‘anaha.
K..
S. ,~nd \‘,~nc\c.
h..
)
T. (1’8
Ti$\uc
.Anti-
Lan and
Wang.
K..
Wony.
Serjeantwn.
.
R.W.S
I l‘jS71J.
S.W
Ilo
C‘han.
Rheumatol
K IA.
Ireland. K.I.
Cohen. N.F.,
,\.CJ..
M ang.
I’..
c1W2) ,\thrllls
I).
Wilson.
D)he\.
\‘ccr-
Rhcum
35. 17’1 D.D.
hla\i.
Talal.
antI
3.
s\.<;..
di Ciovinc.
(lYY2)
\en.
J.H..
(‘hw.
J
Rheumatol.
Llum.
Mol. J.R.,
I‘S,.
I’~~lc&>
D’Alfonso. P.M.
D.M..
t;ktion
60.
11.1’
Ralhcm~~rc.
Grnct.
D.J R.J.
~l’)Shl
Nucluc
Tsa.
(1995) Grant.
Sinnoll.
C.W S.. Stand. S.C.D..
W.J..
-Twt.
J.J. ;IICI
l.lu.
If.%.
14%.
( 1903)
do (ii
Cornpaw). J
S..
Immunul.
I.amb.
P.J. and I Iutchinwl.
I I I3 I I 17.
.tnd hl’l.
-\.l.t
I. 35.:.
22. IJIO
nnd Dulf:
t‘..
P.A..
Mcsl~anc. Winchcstcr.
17::.
h\.G.. de Vi-ies. N.. PocI~~. I. L.B.A.
Dyer.
/CT.. and
16. 1715.
G.W.
Wilson.
J.l..
J.G..
Rheum.
S.A.. Rc\.
E.S.. Fric\.
Schaller.
Arthritis
T‘urncr.
R.. Dcmanlnc. and \‘crgar\i.
h46
E.hl..
,\cid\
WI and Lio.
Dt>-
Bold)rc\a.
.Shingaro\a.
SprlnE.
Marc.
V.(;..
22.
D.G..
Richiardi. (‘hen
,\.M..
21, 342
S.N..
K.. Welsh.
(1981)
M.C’. (1987)
N.S..
H.. Tokunag.
S.. Jimi.
H
[I31 Doherty.
1191 Pociol.
1I.M..
ICot-obukt,.
Ilil.
Putre.
Hochberg.
R.L..
;5.
12. 9 19 Y2i.
B.R..
Dunchle!.
(IYY5)
[2] yeh.
Turc\r\ha!a.
By>tro\.
P. and
\r’.. Hirose.
!g?ns 31. 221
[I51 Miller.
[I]
D.;4..
.,4ntiyn\
c‘.. di C;io\inc.
H.\r’..
S.. :\ito.
Ha\shin\.
S.,I..
K.. Han.
Nishi~ouri.
Rothfield.
References
Iwnberg.
191. Kim.
Mat\uda.
K..
[I81
C’. :2..
(;.I’..
( 19X6)(‘old
L.B.A..
I. Rheumatol.
[I71
R1. and
255.
I 624.
\‘.. Shioha~~a.
[IhI
(I‘~‘)‘)
c1940)Tiysuc
A.h..
Chumakov.
f 199.4) J. Rheumatol. [I’)1 tlashim~,to. I-I.. Twda.
Acknowledgements
h1.J
Georgia.
.\.G..
111tl1101.
[I4
T.M.
K.. Qu~nlan.
6. 247 Warner.
Shakho\.
\1’..4.
ho. 61
Wilson. Putts.
[I II
Bolahrishnan.
Marker?
Filippov.
S.A..
35. 6JI
Lin t’or excellent technical assisFang for preparation of this
L.S..
M.M..
Ovchinniko\.
tl..
I>.. Bachman.
Ma&..
318.
Walport,
S.4..
C‘hu\pilo.
PI
and
A
A.H.L..
and
.Ari7o\.
brynin.
PI
I_.. HotwIt/.
Walker.
137.
[131 Tan.
We thank (‘hia-Hui tance and Ai-Hwei manuscript.
Beischet. Fielder.
Bat&lot-.
A., Wcinrib.
35, il I
C’.D. (19x81 Dis.
[_‘I So. .A.K.L..
dilf‘erencc.
P..
Rheum.
[J] Welch. West.
TNF
D.. Escalante.
Rol-Burman.
1 S . Vati dct 1'7. 557
Scar/a.
47. 501
W.R.. I.\’
R.
,~nd
511-1.
t3rcnchIc~. (l+)S)
Ml.
I’ L.C’..
Transplaw