Download Association of CTLA‐4 but not CD28 gene polymorphisms with

Rheumatology 2001;40:662±667
Association of CTLA-4 but not CD28 gene
polymorphisms with systemic lupus
erythematosus in the Japanese population
S. Ahmed, K. Ihara, S. Kanemitsu, H. Nakashima1, T. Otsuka1,
K. Tsuzaka2, T. Takeuchi2 and T. Hara
Departments of Pediatrics and 1Medicine and Biosystemic Science,
Graduate School of Medical Sciences, Kyushu University, Fukuoka, and
2
Department of Internal Medicine II, Saitama Medical Center, Saitama, Japan
Abstract
Objective. Systemic lupus erythematosus (SLE) in a multisystem autoimmune disorder
characterized by multiorgan pathology and autoantibodies against a variety of autoantigens.
The CD28 and CTLA-4 genes might be candidate genes for SLE, because costimulation signals
from CD80uCD86 to CD28uCTLA-4 have been suggested to play an important role in the
activation or inactivation of T lymphocytes.
Methods. We investigated three polymorphic regions within the CTLA-4 gene, a CuT base
exchange in the promoter region 2318 (CTLA-4 2318CuT), an AuG substitution in the
exon 1 position 49 (CTLA-4 49AuG), an (AT)n repeat polymorphism in the 39 untranslated
region of exon 4 wCTLA-4 39 (AT)nx, and a CD28 gene polymorphism, a TuC substitution in
the intron 3 position +17 (CD28 IVS3 +17TuC), in SLE patients and controls.
Results. SLE patients had signi®cantly higher frequencies of the CTLA-4 49G allele
(P = 0.003) and of the CTLA-4 (AT)n 106 bp allele (P = 0.0008) than controls. We also found
a strong linkage disequilibrium between the A allele of CTLA-4 49AuG and the 86 bp allele
of CTLA-4 39 (AT)n. On the contrary, no association was found between SLE and CTLA-4
2318CuT or CD28 IVS3 +17TuC.
Conclusion. We conclude that the CTLA-4 gene appears to play a signi®cant role in the
development of SLE in the Japanese population.
KEY WORDS: CTLA-4 gene, CD28 gene, Polymorphism, Systemic lupus erythematosus.
Systemic lupus erythematosus (SLE) is an autoimmune
multisystem disorder characterized by the production
of immunoglobulin G autoantibodies. Inappropriate
T-cell-dependent expansion of autoreactive B cells is
considered to play a role in the production of pathogenic autoantibodies against nuclear, cytoplasmic and
cell-surface autoantigens w1x.
T-cell activation requires two discrete signals: a signal
delivered by the T-cell receptor and an accessory signal
that occurs when costimulatory receptors interact with
their ligands. CD28, a major costimulatory molecule,
binds to CD80uCD86 on antigen-presenting cells and
delivers a potent costimulatory signal to T cells w2x.
CTLA-4, a related receptor of CD28, also binds to
CD80uCD86 on antigen-presenting cells but delivers
negative signals to T cells, depending on both the T-cell
activation state and the strength of the T-cell receptor
signal w3x. Thus, CD28 and CTLA-4 molecules regulate
the immune responses to self and foreign antigens by
controlling antigen-speci®c T-cell activation w4x.
The chromosome 2q33 region, where the CTLA-4 and
CD28 genes are located w5x, is one of the potential
susceptibility loci for human SLE w6, 7x. Manipulation
of CD28uCTLA-4 in animal models of autoimmunity
has shown that CD28 as well as CTLA-4 plays a role in
the development of autoimmune disorders w8±10x. In fact,
association studies have revealed that CTLA-4 gene polymorphism is genetically linked to several autoimmune
diseases w11±16x. However, there have been con¯icting
results as to the association between the CTLA-4 gene
and SLE by the examination of a single polymorphism
and there are no data on the association between
the CD28 gene and SLE.
The purpose of this study was to determine which
of the two T-cell costimulatory molecule genes at 2q33
Submitted 5 April 2000; revised version accepted 14 December 2000.
Correspondence to: K. Ihara, Department of Pediatrics, Graduate
School of Medical Sciences, Kyushu University, 3-1-1 Maidashi,
Higashi-ku, Fukuoka 812-8582, Japan.
662
ß 2001 British Society for Rheumatology
Association of CTLA-4 with systemic lupus erythematosus
is linked to the development of SLE by the analysis of
all the known polymorphisms of the CTLA-4 and
CD28 genes.
Materials and methods
Patients and controls
The study population comprised 113 SLE patients.
All these patients ful®lled the American College of
Rheumatology 1982 revised criteria for SLE. The mean
(S.D.) age at onset of SLE was 31 (13) yr. Two hundred
normal individuals (104 males and 96 females) in
the northern Kyushu area of Japan were recruited
for the control population. Informed consent was
obtained from subjects anduor their parents.
DNA extraction
Genomic DNA was obtained from peripheral blood
lymphocytes using the QIAamp DNA extraction kit
(Qiagen, Tokyo, Japan).
Restriction fragment length polymorphism (RFLP)
analysis of CTLA-4 promoter polymorphism
To amplify the region containing the CuT polymorphism
at position 2318, the following primer pairs were used:
forward 59-AATGAATTGGACTGGATGG-39 and
reverse 59-TTACGAGAAAGGAAGCCGTG-39. The
polymerase chain reaction (PCR) was carried out in
a volume of 50 ml containing 40 ng of genomic DNA,
25 pmol of each primer, 1.25 U of Taq polymerase
(Promega, Madison, WI, USA) and 0.2 mM of each
deoxynucleoside triphosphate. The PCR pro®le was as
follows: initial denaturation at 948C for 2 min, followed
by 40 cycles of 948C for 30 s, 608C for 30 s and 728C for
30 s, with ®nal extension at 728C for 7 min. The reaction
products were analysed on 3% agarose gels. To screen
these substitutions, the products were incubated with
MseI restriction enzyme at 378C for 3 h, separated on
FIG. 1. Genotyping of the CTLA-4 gene promoter 2318CuT
polymorphism by MseI RFLP. M, DNA size marker (100-bp
ladder). PCR fragments containing 2318 C are digested
into two fragments (226, 21 bp), whereas PCR fragments
containing 2318 T are digested into three fragments
(21, 96, 130 bp). The 21-bp fragments are not visible on
the agarose gel.
663
3.0% agarose gels and visualized by ethidium bromide
staining (Fig. 1).
Single-strand conformation polymorphism
analysis of CTLA-4 exon 1 49 AuG polymorphism
To amplify the region containing the exon 1 49AuG
polymorphism, the following primer pairs were used:
forward 59-GTTCAAACACATTTCAAAGCTTC-39
and reverse 59-AAATGACTGCCCTTGACTGC-39.
The PCR pro®le was identical to that described above
except for the annealing temperature of 558C and
genomic DNA quantity of 20 ng. For genotype screening, single-strand conformation polymorphism (SSCP)
analysis was carried out with GeneGel Excel 12.5u24
(Amersham Pharmacia Biotech, Uppsala, Sweden)
with 25 mA at 208C, according to the manufacturer's
instructions. Single-strand DNA fragments in the gel
were visualized by subsequent silver staining (Fig. 2).
The results of SSCP analysis were con®rmed by the
direct sequencing of 20 randomly chosen samples.
CTLA-4 39 (AT)n genotype using PCR and
a ¯uorescence-based technique
The CTLA-4 39 untranslated region (UTR) containing
the (AT)n repeat was ampli®ed with the following
primer pairs: forward 59-GCCAGTGATGCTAAAGGTTG-39 and reverse 59-AACATACGTGGCTCTATGCA-39. The 59 end of the forward primer was labelled
with 6-carboxy¯uorescein dye. PCR was employed in a
volume of 25 ml containing 20 ng of genomic DNA,
10 pmol of each primer, 0.625 U of Taq DNA polymerase and 0.2 mM of each deoxynucleoside triphosphate. The PCR conditions were the same as described
above except for the cycles of 30, the annealing
temperature of 558C and the extension time of 15 s.
Genotyping was performed in a mixture of ampli®ed
products with an internal size standard (Gene Scan
2350) by an ABI Prism 310 genetic analyser (PerkinElmer, Foster City, CA, USA).
Allele-speci®c PCR and RFLP analysis of the
polymorphism at position IVS3 +17 of the CD28 gene
We have recently reported a CD28 gene TuC polymorphism in the intron 3 position +17 (CD28 IVS3
+17TuC) w17x. The polymorphism was determined by
FIG. 2. Genotyping of CTLA-4 gene exon 1 49AuG polymorphism by SSCP analysis. Alleles with G and A polymorphisms at nucleotide position 49 show different mobility in
the gels. Lane 1, GuG genotype; lane 2, AuG genotype; lane 3,
AuA genotype.
664
S. Ahmed et al.
(a)
(b)
FIG. 3. Genotype analysis of CD28 IVS3 +17TuC. (a) ASPCR analysis of CD28 IVS3 +17TuC genotype. Lanes 1 and 2, TT, TuT
genotype; lanes 3 and 4, TC, TuC genotype; Lanes 5 and 6, CC, CuC genotype. (b) RFLP analysis by Eco47III of the CD28 IVS3
+17 TuC genotype. PCR fragments containing T are digested into two fragments (126 and 22 bp), whereas PCR fragments with
the CuC genotype are undigested. The 22-bp fragments are not visible on the agarose gel.
allele-speci®c PCR (ASPCR) using an allele-speci®c
primer for C or T at position IVS3 +17 in the CD28
gene. The primers used to detect T and C alleles were
59-CTGGGTAAGAGAAGCAGCAAT-39 (T primer)
and 59-CTGGGTAAGAGAAGCAGCAAC-39 (C
primer) respectively and the common primer 59-CTCAATGCCTTCTGGAAATC-39 (Cm primer). A singlebase mismatch was introduced at position 2 from
the 39 end of both allele-speci®c primers (shown by the
underline). Each primer combination detected only
the primer-speci®c allele (Fig. 3a). To con®rm the accuracy of ASPCR, 91 samples were analysed by restriction
fragment length polymorphism (RFLP). To amplify
the region containing the CD28 IVS3 +17TuC polymorphism, the following primer pairs were used for
the PCR reaction: forward 59-TTTTCTGGGTAAGAGAAGCAGCGC-39 and reverse 59-GAACCTACTCAAGCATGGGG-39. The PCR products were then
incubated with the Eco47III restriction enzyme at 378C
overnight, separated on 3.0% agarose gels and visualized
by ethidium bromide staining (Fig. 3b).
Statistical analysis
Differences between allele or genotype frequencies of
groups were evaluated by x2 analysis with 2 3 2 and
2 3 3 contingency tables with Stat View J-5.0 (software
for Apple Macintosh). When at least one cell number
was not more than 5, Yates' correction was applied to
the x2 value. A P value < 0.05 was considered to be
statistically signi®cant for the CTLA-4 gene 2 318CuT,
49AuG and CD28 gene I VS3 +17TuC analyses. Because
of the multiple comparisons for the microsatellite allele
frequencies, a Bonferroni multiple adjustment was made
to the level of signi®cance in the 39-UTR of the CTLA-4
gene, which was set at P < 0.0045 (0.05u11). The sample
size was suf®cient to detect an odds ratio (OR) of 1.7 or
greater with 80% power at the 5% level of signi®cance,
assuming a frequency of about 50% for the G allele
of the CTLA-4 49AuG polymorphism in the control
population.
Results
Allele and genotype frequencies of CTLA-4 49AuG,
39 (AT)n and 2318CuT
With respect to CTLA-4 gene 49AuG polymorphism,
the frequency of the G allele was signi®cantly higher
in SLE patients than in control subjects (69.5 vs 57.2%,
P = 0.003) because of a signi®cant increase in the frequency of the GG genotype in SLE patients (48.7 vs
31.0%) (Table 1). Regarding the CTLA-4 39 (AT)n
polymorphism, 15 discrete alleles were identi®ed in
the Japanese population with sizes ranging from 86 to
132 base pairs (bp), of which the 86-bp allele was
the most frequent in the control subjects. Compared
with the frequency of the 86-bp allele, the frequency of
the 106-bp allele was signi®cantly higher in SLE patients
than in controls (OR 3.19, P = 0.0008) (Table 2).
There was signi®cant linkage disequilibrium between
the 86-bp allele of the 39 microsatellite polymorphism
and the A allele of the 49AuG polymorphism in exon 1
of the CTLA-4 gene (Table 3). On the other hand, there
were no signi®cant differences in the allele and genotype
frequencies of the 2 318CuT polymorphism between
SLE patients and controls (Table 1).
Allele and genotype frequencies of
CD28 IVS3 +17TuC
No signi®cant differences were observed in the allele
and genotype frequencies of this polymorphism between
SLE patients and control subjects (Table 1).
Discussion
Multiple genetic and environmental factors are involved
in the pathogenesis of SLE w18x. The genetic factors of
SLE include major histocompatibility complex (MHC)
class II genes w19±23x and non-MHC genes, including
genes for complement components, Fc receptor IIuIII,
the T-cell receptor, apoptosis (Fas, Fas-ligand, bcl-2)
and cytokines w24±30x. A recent genome-wide search
Association of CTLA-4 with systemic lupus erythematosus
665
TABLE 1. Comparison of genotype distributions and allelic frequencies between SLE patients and controls
Genotype
CTLA-4 2318CuT
CC
CT
TT
CTLA-4 49AuG
GG
GA
AA
CD28 IVS3 +17TuC
TT
TC
CC
SLE patients:
number (%)
Controls:
number (%)
P
Allele
SLE patients:
number (%)
Controls:
number (%)
95 (84.1)
18 (15.9)
0 (0)
157 (78.5)
43 (21.5)
0 (0)
0.232
C
T
208 (92.0)
18 (8.0)
357 (89.3)
43 (10.7)
0.26
1.39 (0.78±2.47)
55 (48.7)
47 (41.6)
11 (9.7)
62 (31.0)
105 (52.5)
33 (16.5)
0.006
G
A
157 (69.5)
69 (30.5)
229 (57.2)
171 (42.8)
0.003
1.72 (1.20±2.40)
104 (92.0)
8 (7.1)
1 (0.9)
180 (90.0)
18 (9.0)
2 (1.0)
0.834
T
C
216 (95.6)
10 (4.4)
378 (94.5)
22 (5.5)
0.557
1.26 (0.58±2.70)
P
OR (95% CI)
CI, con®dence interval.
TABLE 2. Allele frequencies of respective repeat number polymorphism at the 39 UTR of the CTLA-4 gene in SLE patients and controls
Allele (bp)
86
102
104
106
108
110
112
116
118
120
122
124
126
128
132
Controls (n=400)
130 (32.5)
69 (17.3)
110 (27.5)
18 (4.5)
28 (7.0)
2 (0.5)
0 (0)
1 (0.3)
4 (1.0)
4 (1.0)
15 (3.8)
4 (1.0)
9 (2.3)
5 (1.3)
1 (0.3)
SLE (n=226)
52
44
74
23
9
2
1
0
1
1
7
3
8
1
0
(23.0)
(19.5)
(32.8)
(10.2)
(4.0)
(0.9)
(0.4)
(0)
(0.4)
(0.4)
(3.1)
(1.4)
(3.5)
(0.4)
(0)
OR
95% CI
P
1.00
1.59
1.68
3.19
0.80
2.50
±
±
0.63
0.63
1.17
1.88
2.22
0.50
±
±
0.94, 2.70
1.06, 2.67
1.50, 6.82
0.31, 1.91
0.18, 35.1
±
±
0.01, 6.53
0.01, 6.53
0.38, 3.25
0.26, 11.5
0.70, 6.86
0.01, 4.64
±
±
0.065
0.020
0.0008a
0.599
0.706
±
±
1.000
1.000
0.751
0.694
0.112
0.859
±
Numbers in parentheses are percentages of each allele.
The most common allele (86 bp) in the control subject was chosen as the baseline allele. Each allele of the CTLA-4 gene was compared with the
baseline allele. Shown are the OR and 95% con®dence intervals (CI) for the comparison of allele frequencies between SLE patients and controls.
P was calculated by x2 with a 2 3 2 contingency table.
a
Signi®cant at the 0.0045 level after Bonferroni adjustment to control for type 1 error across multiple comparisons within a locus.
for potential susceptibility loci for human SLE revealed
numerous loci, including 2q33, depending on racial
origin w7x.
The CTLA-4 and CD28 molecules encoded at 2q33
perform critical roles in the regulation of the antigenspeci®c immune response w31x. In the present study,
CTLA-4 but not CD28 gene polymorphisms were
associated with the development of SLE, suggesting
that the genetic factors of SLE are distinct from those of
insulin-dependent diabetes mellitus (IDDM), to which
CD28 gene polymorphism has also been marginally
linked wK. Ihara et al., submitted for publicationx. It is
worthy of note that neither CTLA-4 nor CD28 gene
polymorphisms were associated with atopic asthma,
in a population with the same genetic background w17x.
With respect to the CTLA-4 gene, lack of association
with SLE has been reported in Mexican American,
British, Italian and Japanese populations w30, 32±34x,
while there has been one association reported in
the Slovak population w35x. The studies described
TABLE 3. Linkage disequilibrium between the CTLA-4 49AuG and
CTLA-4 39 (AT)n polymorphisms
CTLA-4 49AuG
CTLA-4 39 (AT)n
AA+GA
GG (without A allele)
With 86-bp allele
Without 86-bp allele
150
45
4
114
P < 0.0001
2
P was calculated by x with a 2 3 2 contingency table.
above were done by the analysis of either the 49AuG
or the 39 (AT)n polymorphism of the CTLA-4 gene. To
increase the reliability of the analysis, we examined all
three polymorphic sites, 49AuG, 39 (AT)n and the 2318
promoter region, and found that the former two
polymorphisms were signi®cantly associated with SLE.
Possible reasons for the con¯icting results might include
the differences in the ethnic groups and the age at
666
S. Ahmed et al.
onset of the disease in the Mexican American, British
and Italian populations and a smaller sample size, as
well as a minor difference in the genetic background in
the Japanese population. As has been found for IDDM,
Graves disease, Hashimoto's thyroiditis, multiple sclerosis and rheumatoid arthritis w11±15, 36, 37x, the G allele
of the CTLA-4 49AuG polymorphism was associated
with SLE in recent studies w35x and the present work.
The 106-bp allele of the CTLA-4 39 (AT)n polymorphism showed a signi®cant association with SLE,
consistent with results from other autoimmune disorders
such as IDDM, Graves disease, Addison's disease and
Wegener's granulomatosis w12, 13, 16, 38x.
The CTLA-4 49AuG and 39 (AT)n polymorphisms are
unlikely to affect the function of the gene because
49AuG and 39 (AT)n are located on the peptide leader
and the 39 untranscribed region respectively. However, it
is possible that either or both of the CTLA-4 gene
polymorphisms affects CTLA-4 mRNA stability and
subsequent CTLA-4 expression w11x. CTLA-4-de®cient
mice develop a lethal lymphoproliferative disease by
massive uncontrolled T cells w8, 9x. CTLA-4 not only
counterbalances CD28 signals but also inhibits T-cell
responses independently of CD28. Recent studies have
suggested that CTLA-4 interacts with the CD3z chain of
the T-cell receptor complex and interferes with very
early T-cell receptor signalling events w39x. Thus, CTLA-4
might induce autoimmunity by the inhibiting CD3z
signal because a decrease in CD3z expression was
associated with SLE in a proportion of cases w40x.
In conclusion, the CTLA-4 gene appears to play
a signi®cant role in the development of SLE in Japanese
population. Further study will be necessary to elucidate
the mechanisms of the association between CTLA-4
gene polymorphisms and SLE.
Acknowledgements
We extend special thanks to Yoko Katafuchi, Tamami
Tanaka and Kanako Uchida for technical assistance.
This work was supported by Grant-in-Aid for Scienti®c
Research (B) from the Ministry of Health and Welfare
and Ministry of Education, Science, and Culture of
Japan.
References
1. Kotzin BL. Systemic lupus erythematosus. Cell 1996;
85:303±6.
2. Lenschow DJ, Walunas TL, Bluestone JA. CD28uB7
system of T cell costimulation. Annu Rev Immunol
1996;14:233±58.
3. Scheipers P, Reiser H. Role of the CTLA-4 receptor
in T cell activation and immunity. Physiologic function of the CTLA-4 receptor. Immunol Res 1998;18:
103±15.
4. Oosterwegel MA, Greenwald RJ, Mandelbrot DA,
Lorsbach RB, Sharpe AH. CTLA-4 and T cell activation.
Curr Opin Immunol 1999;11:294±300.
5. Lafage-Pochitaloff M, Costello R, Couez D et al. Human
CD28 and CTLA-4 Ig superfamily genes are located
on chromosome 2 at bands q33±q34. Immunogenetics
1990;31:198±201.
6. Moser KL, Neas BR, Salmon JE et al. Genome scan of
human systemic lupus erythematosus: evidence for linkage
on chromosome 1q in African-American pedigrees. Proc
Natl Acad Sci USA 1998;95:14869±74.
7. Gaffney PM, Kearns GM, Shark KB et al. A genome-wide
search for susceptibility genes in human systemic lupus
erythematosus sib-pair families. Proc Natl Acad Sci USA
1998;95:14875±9.
8. Tivol EA, Borriello F, Schweitzer AN, Lynch WP,
Bluestone JA, Sharpe AH. Loss of CTLA-4 leads to
massive lymphoproliferation and fatal multiorgan tissue
destruction, revealing a critical negative regulatory role of
CTLA-4. Immunity 1995;3:541±7.
9. Waterhouse P, Penninger JM, Timms E et al.
Lymphoproliferative disorders with early lethality in
mice de®cient in Ctla-4. Science 1995;270:985±8.
10. Herold KC, Vezys V, Koons A, Lenschow D,
Thompson C, Bluestone JA. CD28uB7 costimulation
regulates autoimmune diabetes induced with multiple low
doses of streptozotocin. J Immunol 1997;158:984±91.
11. Nistico L, Buzzetti R, Pritchard LE et al. The CTLA-4
gene region of chromosome 2q33 is linked to, and
associated with, type 1 diabetes. Hum Mol Genet 1996;
5:1075±80.
12. Marron MP, Raffel LJ, Garchon HJ et al. Insulindependent diabetes mellitus (IDDM) is associated with
CTLA-4 polymorphisms in multiple ethnic groups.
Hum Mol Genet 1997;6:1275±82.
13. Yanagawa T, Hidaka Y, Guimaraes V, Soliman M,
DeGroot LJ. CTLA-4 gene polymorphism associated
with Graves' disease in a Caucasian population. J Clin
Endocrinol Metab 1995;80:41±5.
14. Donner H, Rau H, Wal®sh PG et al. CTLA4 alanine-17
confers genetic susceptibility to Graves' disease and
to type 1 diabetes mellitus. J Clin Endocrinol Metab
1997;82:143±6.
15. Donner H, Braun J, Seidl C et al. Codon 17 polymorphism
of the cytotoxic T lymphocyte antigen 4 gene in
Hashimoto's thyroiditis and Addison's disease. J Clin
Endocrinol Metab 1997;82:4130±2.
16. Huang D, Giscombe R, Zhou Y, Karilefvert A. Polymorphisms in CTLA-4 but not tumor necrosis factor-a or
interleukin 1b genes are associated with Wegener's
granulomatosis. J Rheumatol 2000;27:397±401.
17. Nakao F, Ihara K, Ahmed S et al. Lack of association
between CD28uCTLA-4 gene polymorphisms and atopic
asthma in Japanese population. Exp Clin Immunogenet
2000;17:179±84.
18. Vyse TJ, Kotzin BL. Genetic susceptibility to systemic
lupus erythematosus. Annu Rev Immunol 1998;
16:261±92.
19. Hochberg MC, Peteri M, Machan C, Bias WB. HLA
class II alleles DRB1*1501u15.3 and DQB1*0602 are
associated with systemic lupus erythematosus (SLE) in
African-Americans. Arthritis Rheum 1991;34:S140.
20. Hashimoto H, Nishimura Y, Dong RP et al. HLA
antigens in Japanese patients with systemic lupus erythematosus. Scand J Rheumatol 1994;23:191±6.
21. Bell DA, Maddison PJ. Serologic subsets in systemic lupus
erythematosus: an examination of autoantibodies in
relationship to clinical features of disease and HLA
antigens. Arthritis Rheum 1980;23:1268±73.
Association of CTLA-4 with systemic lupus erythematosus
22. Reveille JD. The molecular genetics of systemic lupus
erythematosus and SjoÈgren's syndrome. Curr Opin
Rheumatol 1992;4:644±56.
23. Olsen ML, Arnett FC, Reveille JD. Contrasting molecular patterns of MHC class II alleles associated with
the anti-Sm and anti-RNP precipitin autoantibodies in
systemic lupus erythematosus. Arthritis Rheum 1993;
36:94±104.
24. Walport MJ, Lachmann PJ. Complement de®ciencies and
abnormalities of the complement system in systemic
lupus erythematosus and related disorders. Curr Opin
Rheumatol 1990;2:661±3.
25. Davies EJ, Snowden N, Hillarby MC et al. Mannosebinding protein gene polymorphism in systemic lupus
erythematosus. Arthritis Rheum 1995;38:110±4.
26. Salmon JE, Millard S, Schachter LA et al. Fc gamma
RIIA alleles are heritable risk factors for lupus nephritis in
African Americans. J Clin Invest 1996;97:1348±54.
27. Duits AJ, Bootsma H, Derksen RH et al. Skewed
distribution of IgG Fc receptor IIa (CD32) polymorphism
is associated with renal disease in systemic lupus erythematosus patients. Arthritis Rheum 1995;38:1832±6.
28. Haseley LA, Wisnieski JJ, Denburg MR et al. Antibodies
to C1q in systemic lupus erythematosus: characteristics
and relation to Fc gamma RIIA alleles. Kidney Int 1997;
52:1375±80.
29. Wu J, Wilson J, He J, Xiang L, Schur PH, Mountz JD.
Fas ligand mutation in a patient with systemic lupus
erythematosus and lymphoproliferative disease. J Clin
Invest 1996;98:1107±13.
30. Mehrian R, Quismorio FP Jr, Strassmann G et al.
Synergistic effect between IL-10 and bcl-2 genotypes in
determining susceptibility to systemic lupus erythematosus. Arthritis Rheum 1998;41:596±602.
31. Anderson DE, Bieganowska KD, Bar-Or A et al.
Paradoxical inhibition of T-cell function in response to
CTLA-4 blockade: heterogeneity within the human T-cell
population. Nature Med 2000;6:211±4.
667
32. Heward J, Gordon C, Allahabadia A, Barnett AH,
Franklyn JA, Gough SC. The A±G polymorphism
in exon 1 of the CTLA-4 gene is not associated with
systemic lupus erythematosus. Ann Rheum Dis 1999;
58:193±5.
33. D'Alfonso S, Rampi M, Bocchio D, Colombo G, ScorzaSmeraldi R, Momigliano-Richiardi P. Systemic lupus
erythematosus candidate genes in the Italian population.
Arthritis Rheum 2000;43:120±8.
34. Matsushita M, Tsuchiya N, Shiota M et al. Lack of
a strong association of CTLA-4 exon 1 polymorphism
with the susceptibility to rheumatoid arthritis and systemic
lupus erythematosus in Japanese: an association study
using a novel variation screening method. Tissue Antigens
1999;54:578±84.
35. Pullmann R, Lukac J, Skerenova M et al. Cytotoxic
T lymphocyte antigen 4 (CTLA-4) dimorphism in patients
with systemic lupus erythematosus. Clin Exp Rheumatol
1999;17:725±9.
36. Ligers A, Xu C, Saarinen S, Hillert J, Olerup O.
The CTLA-4 gene is associated with multiple sclerosis.
J Neuroimmunol 1999;97:182±90.
37. Gonzalez-Escribano MF, Rodriguez R, Valenzuela A,
Garcia A, Garcia-Lozano JR, Nunez-Roldan A. CTLA 4
polymorphisms in Spanish patients with rheumatoid
arthritis. Tissue Antigens 1999;53:296±300.
38. Kemp EH, Ajjan RA, Husebye ES et al. A cytotoxic T
lymphocyte antigen-4 (CTLA-4) gene polymorphism is
associated with autoimmune Addison's disease in English
patients. Clin Endocrinol 1998;49:609±13.
39. Lee KM, Chuang E, Grif®n M et al. Molecular basis
of T cell inactivation by CTLA-4. Science 1998;
282:2263±6.
40. Liossis SN, Ding XZ, Dennis GJ, Tsokos GC. Altered
pattern of TCRuCD3-mediated protein-tyrosyl phosphorylation in T cells from patients with systemic lupus
erythematosus. De®cient expression of the T cell receptor
zeta chain. J Clin Invest 1998;101:1448±57.