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Major histocompatibility complex class ii gene associations with antiu1 small nuclear ribonucleoprotein antibody relationship to immunoreactivity with individual constituent proteins.

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ARTHRITIS & RHEUMATISM Volume 38
Number 3, March 1995. pp 396-405
0 1995, American College of Rheumatology
396
MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I1 GENE
ASSOCIATIONS WITH ANTI-U1 SMALL NUCLEAR
RIBONUCLEOPROTEIN ANTIBODY
Relationship t o Immunoreactivity with Individual Constituent Proteins
MASATAKA KUWANA, Y UTAKA OKANO, JUNICHI KABURAKI,
KIMIYOSHI TSUJI, and HIDETOSHI INOKO
Objective. To better define immunogenetic associations with the anti-U1 ribonucleoprotein (U1 RNP)
autoantibody response.
Methods. HLA class I1 alleles were determined by
genotyping in 49 Japanese rheumatic disease patients
with anti-U1 RNP antibody and 43 race-matched
healthy controls. Immunoreactivities to U1 RNP constituent proteins (70K, A, B/IB', and C) were detected by
immunoblots using purified HeLa cell Sm antigen, and
antibody titer was determined by passive hemagglutination assay.
Results. DQB1*030:1 was significantly more frequent in anti-Ul RNP-positive patients than in controls
(43% versus 14%; odds ratio [OR] = 4.6, corrected P =
0.03). All anti-U1 RNP-positive patients had either a
DQBl"0601, *0602, *03011, *0302, or "0303 allele,
which share tyrosine at potsition 30, and the amino acid
sequence Thr, Arg, Ala, Glu, Leu, Asp, and Thr at
positions 71-77 in the DQBl pl domain. In contrast,
one of these alleles was found in 81% of the controls
Supported in part by the Scleroderma Grant for Intractable
Disease from the Japanese Mirtistry of Health and Welfare, and
grants-in-aid from the Ministry 01- Education, Science and Culture of
Japan.
Masataka Kuwana, MD: Keio University School of Medicine, Tokyo, Japan (current ahddress: University of Pittsburgh
School of Medicine, Pittsburgh, Pennsylvania); Yutaka Okano, MD:
Nippon Kokan Hospital, Kawasaki, Japan; Junichi Kaburaki, MD:
Keio University School of Medicine: Kimiyoshi Tsuji, PhD, Hidetoshi Inoko, PhD: Tokai University School of Medicine, Isehara,
Japan.
Address reprint requests to Masataka Kuwana, MD, Division of Rheumatology and Clinical Immunology, Department of
Medicine, University of Pittsburgh School of Medicine, El 109
Biomedical Science Tower, Pittsburgh, PA 15213.
Submitted for publication June 27, 1994; accepted in revised form October 5 , 1994.
(OR = 24, P = 0.002). In addition, anti-U1 RNP
antibody was associated with unique DQBl*O302;
DRB1*0401 haplotype. Anti-70K reactivity and antibody titer were positively associated with a basic amino
acid residue, arginine or histidine, at position 13 (DR2
or DR4) and were negatively associated with the amino
acid sequence Ile, Leu, Glu, and Asp at positions 67-70,
which was present in some of the DR5-, DR6-, and
DRS-associated alleles, in the DRBl pl domain. Anti-C
reactivity was strongly associated with DR2, particularly with DRB1*1502.
Conclusion. The several shared epitopes located
on HLA-DRB1 and DQBl genes control the anti-U1
RNP autoantibody response.
A striking feature of many autoimmune diseases is their association with certain genes of the
major histocompatibility complex (MHC) region ( 1).
Recent molecular analyses of genes encoding HLA
class I1 molecules have revealed that certain antinuclear antibodies (ANA) are associated with specific
amino acid residue(s) of the MHC molecule. For
example, associations of anticentromere antibody with
a polar amino acid residue at position 26 of the DQB 1
domain ( 2 ) and anti-Ro/SS-A antibody with the combination of glutamine at position 34 in the DQAl
domain and leucine at position 26 in the DQBl domain
(3) have been reported. Recently, we have reported a
strong association between anti-DNA topoisomerase I
antibody and the combination of HLA-DRB and
DQBl genes in Japanese patients with systemic sclerosis (SSc) (4). Since the HLA class I1 molecule
presents processed antigen to the helper T cells,
resulting in their activation, proliferation, and induc-
MHC CLASS I1 GENE ASSOCIATIONS WITH ANTI-Ul RNP
tion of effector function (5,6), these ANA and HLA
class I1 associations suggest that ANA production is
genetically mediated by an interaction between HLA
class I1 molecules and autoantigen-reactive T cells.
The U series of small nuclear ribonucleoprotein
(snRNP) particles, including U1, U2, U4/U6, and U5,
are major targets of autoimmunity in rheumatic diseases (7). A high titer of anti-U1 RNP antibody was
first described as a serologic marker for mixed connective tissue disease (MCTD), with shared clinical
features of systemic lupus erythernatosus (SLE), SSc,
and polymyositis (8). However, anti-U1 RNP antibody was also detected in sera from patients with S L E
or SSc alone, as well as those who had only Raynaud’s
phenomenon. Anti-U 1 RNP antibody in patients’ sera
has been shown to recognize the constituent proteins
of molecular weight 70,000 (70K), 33,000 (A), 29,000
(B’), 28,000 (B), and 22,000 (C) (9). Immunoreactivities to B and B’ proteins are simultaneous, but those to
the 70K, A, B/B’, and C proteins are immunologically
independent (9-1 1). These observations indicate that
patients with anti-U1 R N P antibody are heterogeneous, both clinically and serologically.
Several studies of immunogenetic correlations
with anti-U 1 RNP antibody employing serologic HLA
typing have shown an increased frequency of DR4 in
white patients (12-16) and of DQ3 in Japanese patients
(17), but some investigators failed to detect any associations (18,19). Recent molecular immunogenetic analysis has revealed an increased frequency of one of the
DQ3 subantigens, DQ8 (DQB1*0302), in white patients with S L E and anti-U1 RNP antibody, as compared with race-matched healthy controls (20). Another study demonstrated that H L A class I1 allele
conferring susceptibility to MCTD was the HLADRB1*0401 ;DRB4*0101 ;DQA1*03;DQBl*0301 haplotype in Japanese patients (21). Since these associations were generally weak and sometimes inconsistent
with each other, we hypothesized that they resulted
from serologic heterogeneity in anti-U 1 RNP antibodypositive patients.
In the present study, 49 Japanese patients with
anti-U1 RNP antibody were evaluated for HLA class
I1 alleles by the polymerase chain reaction (PCR)
restriction fragment length polymorphism (RFLP)
method (22-26), in addition to conventional serologic
HLA class I and class I1 typing, in order to detect
HLA associations with both immunoreactivities to
individual U1 RNP proteins and antibody titer.
397
PATIENTS ANlD METHODS
Patients and controls. Forty-nine unrelated Japanese
patients with rheumatic diseases seen for followup evaluation from October 1992 through January 1993 were studied:
45 seen at Keio University School of Medicine (Tokyo) and
4 at Nippon Kokan Hospital (Kawasaki, adjacent to Tokyo).
All patients were previously determined to be positive for
anti-U1 RNP but negative for anti-Sm antibody by double
immunodiffusion tests of sera obtained at their first visit.
Clinical diagnoses were overlap syndrome in 15, SLE in 14,
SSc in 9, and unclassified connective tissue disease in 11.
Fifteen and 24 patients satisfied the MCTD criteria proposed
by Porter et a1 (27) and Alarcon-Segovia and Villarreal (28),
respectively. Twenty-three patients met the American College of Rheumatology (ACR; formerly, the American Rheumatism Association) classification criteria for SLE (291, 24
met the ACR preliminary classification criteria for SSc (30),
and 10 met the diagnostic criteria for definite polymyositis or
dermatomyositis as described by Bohan and Peter (3 1).
Fifteen patients satisfied two or more of these criteria, and
I 1 satisfied none of them.
Race-matched healthy subjects who had no symptoms related to the rheumatic diseases served as controls;
these were 43 Japanese individuals living in the TokyoKawasaki area. Seven sera -From healthy controls were
negative for anti-U 1 RNP antibody by double immunodiffusion tests; sera were not available from the remaining
healthy controls. We believe that all the healthy controls
were negative for anti-Ul RNP antibody, because we had
confirmed that anti-U1 RNP antibody was not detected in
sera from 100 healthy donors in another series. To confirm
HLA haplotypes, we also performed genotyping of HLA
class I1 genes in the family members (parents and/or children) of some of the anti-Ul RNP-positive patients.
Anti-U1 RNP antibody assays. Serum samples were
obtained at the first visit and stored at -20°C until used.
Double immunodiffusion tests were performed using rabbit
thymus extract as an antigen slource (32), and anti-UI RNP
and anti-Sm antibodies were identified according to precipitin lines that were identical to those of the reference sera.
Anti-extractable nuclear antigen (anti-ENA) antibody titer
was determined by standard passive hemagglutination assays in microtiter plates using calf thymus ENA (33), and the
results were recorded as the ainti-U1 RNP antibody titer.
Immunoreactivities to the individual proteins of Ul
RNP (70K, A, BIB’, and C) were determined on immunoblots using purified Sm antigen as an antigen source. The Sm
antigen was purified from HeLa cells by immunoaffinity
chromatography, separated tiy sodium dodecyl sulfatepolyacrylamidegel electrophoresis, and transferred to nitrocellulose membranes (34). Serum samples diluted at 1:250
with Tris buffered saline (0.02M Tris HCI, 140 mM NaCI, pH
7.4) were used as the first antibody. IgG bound to the
proteins was visualized by an enzyme-linked immunoassay
(Promega, Madison, WI). When insufficient signals were
obtained in the standard immunoblots, serum samples diluted at 1: 100 were rechallenged.
HLA class I and class I1 typing. Serologic HLA class
I (A, B, and C) and class I1 (DR and DQ) typing was
performed by standard complement-dependent microlym-
KUWANA ET AL
phocytotoxicity tests (35). For HLA class I1 genotyping,
genomic DNA was isolated from peripheral blood leukocytes (36). The HLA-DRB1, DRB3, DRB4, DQB1, and
DPBl genes were determined by the PCR-RFLP method as
described elsewhere (22-26). For discrimination of the
DQB 1*0602 and DQB 1*0603 alleles, the PCR-amplified
DNA was directly cloned into the M13 sequence vector and
transformed into Escherichia cofi JMlOl . Single-stranded
DNA templates were sequenced by the method of Sanger et
a1 (37).
Statistical analysis. Dlifferences in frequencies were
analyzed by chi-square test, and Fisher’s 2-tailed exact test
was used when the expected frequency in 1 of 4 cells of the
2 x 2 tables was less than 5. Corrected P values (Pco,,) were
obtained by multiplying observed P values by the number of
comparisons made (HLA-DEI x 12, DRBl x 44, DRB3 x 4,
DQBl x 13, and DQB1-DRB1 x 100). The odds ratio (OR)
of the significant differences was calculated. When 1 of the 4
cells of the 2 x 2 tables was 0, Haldane’s modification was
applied (38). Comparisons of anti-ENA antibody titers between patient groups were made using Wilcoxon rank sum
tests.
RESULTS
HLA associations iin anti-U1 RNP antibodypositive patients. We performed serologic typing of
HLA class I (A, B, and C) and class I1 (DR and DQ)
antigens in 40 of 49 patieints with anti-U1 RNP antibody and in 43 normal controls, and genotyping of
HLA class I1 alleles (DR.Bl, DRB3, DRB4, DQB1,
and DPB1) in all patients and normal controls. No
significant differences in tlhe frequencies of any types
of HLA class I antigens or DPBl alleles were observed
between these 2 groups (data not shown).
As shown in Table I , DRB 1*0401, DRB 1*0802,
and DQB 1*0302 were significantly more frequent in
patients with anti-U1 RNP antibody than in normal
controls. In contrast, frelquencies of DR6 (all combined of DRBl*1301, *1302, *1305, *1401, and *1402)
and DQB 1*06W were significantly decreased among
the patients. However, none of these differences
reached statistical significance after correction of the P
values except for DQB1*01302, which was found in 21
(43%) of 49 patients with anti-U1 RNP antibody
compared with 14% of normal controls (P,,, = 0.03).
In the 28 DQB 1*0302-negative patients with
anti-U1 RNP antibody, 18 and 13 patients had
DQB 1*0601 and DQB 1*03103,respectively, and 6 had
both of these alleles. The remaining 3 patients had
either DQB 1*0602 or DQB 1*0301. The DQB 1*0601,
*0602, *0301, *0302, and *lo303 alleles share a tyrosine
residue at position 30 and a 7-amino acid sequence
containing Thr, Arg, Ala, Glu, Leu, Asp, and Thr at
Table 1. Frequencies of HLA-DRBl, DRB3, DRB4, and DQBl
genes in 49 Japanese rheumatic disease patients with anti-U1 RNP
precipitin antibody and 43 normal controls
Patients with
anti-U1 RNP
(n = 49)
Phenotype
Allele
no.
Frequency
DR 1
DR2
DRB 1*0101
All combined
DRB 1 * 1501
DRB1*1502
DRB1*1602
All combined
DRB 1*0401
DRB 1*0403
DRB1*0405
DRB1*0406
DRBI*0407
DRB 1*0410
All combined
DRB 1* 1101
DRB 1* 1 102
DRB 1* 1201
DRBI*1202
All combined
DRB 1 * 1301
DRB1*1302
DRB1*1305
DRB 1* 1401
DRB1*1402
All combined
DRBI*0802
DRBI*0803
DRBl*0901
DRB 1* 1001
All combined
DRB3*0101
DRB3*0202
DRB3*0301
DRB4*0101
DQB1*0501
DQB1*0502
DQB1*0503
DQB1*0601
DQB1*0602
DQB1*0604
DQB1*0301
DQB1*0302
DQB1*0303
DQB1*0401
DQB1*0402
7
20
2
18
2
19
8
1
10
1
1
0.14
0.41
0.04
0.37
0.04
0.39
0.16*
0.02
0.20
0.02
0.02
0.02
0.10
0.02
0.02
0.06
0
0.12t
0.02
0.04
0.04
0.02
0
0.22
0.14t:
0.08
0.33
0
0.43
0.02
0.12
0.08
0.65
DR4
DR5
DR6
DR8
DR9
DRlO
DR52
DR53
DQ 1
DQ3
DQ4
* Odds ratio (OR) = 8.2, P
t OR = 0.29, P = 0.02.
t: OR = 7.0, P = 0.04.
1
5
1
1
3
0
6
I
2
2
1
0
I1
7
4
16
0
21
1
6
4
32
7
3
1
22
2
2
5
21
17
9
2
0.06
0.02
0.45
0.04
0.043
0.10
0.4317
0.35
0.18
0.04
no.
Frequency
5
0.12
0.33
0.12
0.21
0
0.49
0.02
0.07
0.33
0.07
0
0
0.14
0.05
0.02
0.02
0.05
0.33
0
0.14
14
5
9
0
21
1
3
14
3
0
0
6
2
1
1
2
14
0
6
0
5
3
8
1
8
11
2
26
3
13
6
29
8
4
5
17
5
7
8
6
10
10
3
0
0.12
0.07
0.19
0.02
0.19
0.26
0.05
0.60
0.07
0.30
0.14
0.67
0.19
0.09
0.12
0.40
0.12
0.16
0.19
0.14
0.23
0.23
0.07
= 0.03.
§ OR = 0.22, P = 0.04.
= 4.6, P = 0.002 (corrected
11 OR
0.14
Normal controls
(n = 43)
P
=
0.03).
positions 71-77 (Y30 and T71RAELDT77)in the /3l
domain of the DQBl gene. The frequency of this
combination of amino acid residues (100%) was significantly increased in patients with anti-U1 RNP anti-
MHC CLASS I1 GENE ASSOCIATIONS WITH ANTI-Ul RNP
Estimated HLA-DQB 1 *0302;DRB1 haplotype frequencies in 49 Japanese rheumatic disease patients with anti-Ul RNP
precipitin antibody and normal controls
Table 2.
Normal controls
DQB1;DRBI haplotype
DQB 1*0302;
DRB1*0401
DRB 1*0403
DRB 1*0406
DRB 1*0407
DRBI*IIOI
DRBl*1201
DRB1*1305
DRB 1*0802
Patients with
anti-U1 RNP
(n = 98)
Present
study
(n = 86)
no.
HF
no.
HF
HF
7 (5)t
l(1)
0.078
0.01
0.01
0.01
0.01
0.02
0.02
0.061
0
0
0.03
0.03
0
0
0
0
0
<0.007
0.027
0.030
C0.007
<0.007
0.007
<0.007
0.010
1
I
1 (1)
2
2 (1)
6 (4)
3
3
0
0
0
0
0
1Ith HLA
Workshop
(n = 306)*
* Haplotype frequencies (HF) in Japanese normal controls were
obtained from the 11th International Histocompatibility Workshop
and Conference (39).
t Numbers in parentheses show those confirmed by HLA genotyping of family members.
8 Odds ratio (OR) = 14, P = 0.03 compared with normal controls in
present study, and OR > 23, P < O.OOO1 (corrected P < 0.01)
compared with normal controls in the llth HLA Workshop.
5 OR = 6.6, P = 0.009 compared with normal controls in the llth
HLA Workshop.
body compared with that in normal controls (81%;
OR = 24, P = 0.002). This amino acid residue combination is not found in any other known DQBI alleles.
Taken together, these results suggest that the Y30 and
T71RAELDT77sequence in the DQBl pl domain are
essential for anti-U 1 RNP autoantibody production.
Table 2 shows the estimated DQB1*0302;DRB1
haplotype frequencies in patients with anti-U1 RNP
antibody and in normal controls. The DQB1*0302;
DRB 1*0401 and DQB 1"0302;DRB 1*0802 haplotypes
were found in 7 and 6 patients with anti-U1 RNP
antibody, respectively, but in none of 43 normal
controls in this study. In 6 normal control subjects
with DQB 1*0302 we found DRB 1*0403 and
DRB 1*0406 in 3 persons each. We have confirmed the
DQB1*0302;DRB1*0401 haplotype in 5 of the 7 patients and the DQB l *0302;DRB1*0802 haplotype in 4
of the 6 patients by genotyping of their family members.
When we compared the DQBl*0302;DRB 1
haplotype frequencies in anti-U 1 RNP-positive patients (n = 98) with those in healthy Japanese controls
in this study (n = 86) and in the 11th International
Histocompatibility Workshop and Conference (n =
306) (39), the association with the uncommon
DQB 1*0302;DRB1*0401 haplotype was confirmed.
399
Genealogic survey revealed no genetic admixture from
other ethnic groups in patients with the DQB 1*0302;
DRB 1*0401 haplotype. This different DQB 1*0302;
DRB 1 haplotype distribution between patients with
anti-U1 RNP antibody and normal controls suggests
that anti-U1 RNP antibody is not associated with
DQBl*O302 alone, but rather, with the DQB1*0302;
DRB I haplotypes with a unique linkage disequilibrium.
Two additional haplotypes, DQBl*O601;
DRB1*1502 and DQB1*0303;DRB1*0901,characteristic of the Japanese population (39), were also more
frequently found in patients with anti-U1 RNP antibody than in normal controls. Although these 2 differences were not statistically significant in comparison
between all groups, they reached statistical significance in the limited comparison between the 36
anti-U1 RNP-positive patients and the 43 normal
controls who did not have the DQB 1*0302;DRB1*0401
or DQB 1*0302;DRB1*0802 haplotype (44% versus
21%; OR = 3.0, P = 0.03, and 44% and 23%; OR =
2.6, P = 0.04, respectively).
HLA associations wit11 immunoreactivities to individual U1 RNP proteins. In sera from 49 patients with
anti-U1 RNP antibody, 36 (73%), 46 (94%), 43 (88%),
and 31 (63%) recognized the 70K, A, B/B', and C
proteins of U1 RNP, respectively. There were no
significant correlations among the reactivities of these
individual proteins, except for those to BIB' and C
proteins, which showed a negative correlation. Specifically, 70% of anti-B/B'-positive patients had anti-C
reactivity, whereas 17% of anti-B/B' negative patients
had anti-C reactivity (P = 0.02). Next, 49 anti-U1
RNP-positive patients were grouped according to the
presence or absence of immunoreactivity to each
protein, and differences in HLA class I antigen and
class I1 allele frequencies between these 2 groups were
investigated. As a result, significant differences were
obtained for anti-70K and anti-C reactivities, but not
for anti-A or anti-B/B' reactivity. All of these significant differences were found at the HLA-DR and DQ
loci, but not at the HLA class I or HLA-DP locus.
When we compared the 36 anti-70K-positive
and 13 anti-70K-negative patients (Table 3), DR2,
DRB1*1502 (subtype of DR:!), DR4, DRB1*0405 (subtype of DR4), and DQB1*0401 were significantly more
frequent in anti-70K-posil ive patients. Moreover,
DR2 (56%) and DRBl*150;! (50%) were significantly
more frequent in anti-70K--positive patients than in
normal controls (33% and 21%, respectively; OR =
2.6, P = 0.04, and OR = 3.8, P = 0.007, respectively).
KUWANA ET AL
400
Table 3. Frequencies of HLA-DRBI, DRB3, DRB4, and DQBl
genes in 49 Japanese rheumatic disease patients with anti-U1 RNP
antibody, according to the presence or absence of immunoreactivity
to the U1 RNP 70K protein
Phenotype
DR 1
DR2
DR4
DR5
DR6
DR8
DR9
DR52
DR53
DQ 1
DQ3
DQ4
Anti-70K
positive
(n = 36)
Anti-70K
negative
(n = 13)
Allele
no.
Frequency
no.
Frequency
DRB 1*0101
All combined
DRB 1*1501
DRB 1* 1502
DRB 1* 1602
All combined
DRB 1*0401
DRB 1*0403
DRB 1*0405
DRB 1*M06
DRB 1*0407
DRB 1*0410
All combined
DRB 1* 1101
DRBl *I 102
DRB 1* 1201
All combined
DRB 1* 1301
DRB1*1302
DRB 1* 1305
DRB 1* 1401
All combined
DRB I *Of302
DRB 1*0803
DRB1*0901
All combined
DRB3*0101
DRB3*0202
DRB3*0301
DRB4*0101
DQB1*0501
DQB 1*0502
DQB 1*0503
DQB1*0601
DQB 1*0602
DQB 1*0604
DQB 1*0301
DQB 1*0302
DQB 1*0303
DQB1*0401
DQB 1*0402
5;
20
2:
0.14
0.56*
0.06
0.50t
0.06
0.47$
0.22
0.03
0.285
0
2
0
0
0
0
2
0
0
0
0. I5
0
0
18;
2:
17
8
1
10
a1
a)
I
3
1
1
1
1
0'
0'
a
1
6
6
0
11
10
0
4
0
26
5
2
1
18
2
0
4
15
12
9
2
o
0.03
0.08
0.03
0.03
0.03
0.037
0
0
0
0.03
0.17
0.17
O#
0.31
0.28**
0
0.11
Ott
0.72
0.14
0.06
0.03
0.50
0.06
0
0.11
0.42
0.33
0.25$$
0.06
1
1
0
2
0
0
2
5
1
2
2
0
5
1
4
5
11
1
2
4
6
2
1
0
4
0
2
I
6
5
0
0
* Odds ratio (OR) = 34, P = 0.0005 (corrected P [P,,]
t OR = 27, P = 0.002.
0
0
0.15
0
0
0
0.08
0.08
0
0.15
0
0
0.15
0.38
0.08
0.15
0.15
0
0.38
0.08
0.31
0.38
0.85
0.08
0.15
0.31
0.46
0.15
0.08
0
0.31
0
0.15
0.08
0.46
0.38
0
0
= 0.007).
$ OR = 4.1, P = 0.04.
5 OR = 11, P = 0.03.
7 OR = 0.046, P = 0.0008 (P,,, = 0.009).
# OR = 0.032, P = 0.005.
** OR = 0.070, P = 0.006.
tt OR = 0.032, P = 0.005 (P,,, = 0.02).
$$ OR = 9.3, P = 0.04.
The frequency of DRBlW401 (subtype of DR4) was
not significantly increased in anti-70K-positive versus
negative patients (P = 0.06), but was significantly
higher in anti-70K-positive patients versus normal
controls (OR = 8.8, P = 0.007).
DRB 1*0405 and DRB 1*0401 were found exclusively in the anti-70K-positive patient group (n = 10
and n = 8). The association of anti-70K reactivity with
DQB 1*0401 may be explained by the known linkage
disequilibrium of DQB 1*0401 and DRB 1*0405 in the
Japanese population (39). All 20 DRZpositive and 17
of the 19 DRCpositive patients were anti-70K positive,
and either DR2 or DR4 was found in 86% of the
anti-70K-positive patients but in only 15% of the
anti-70K-negative patients (OR = 34, P = 0.00002)
and in 65% of the normal controls (OR = 3.3, P =
0.02). All known DR2- and DR4-associated alleles
share a basic amino acid residue, arginine or histidine,
at position 13 in the first hypervariable region of the
DRBl pl domain, in contrast to a neutral-charged
amino acid residue in the other known DRBl alleles.
Therefore, this charge difference at position 13 seems
to be important for anti-70K reactivity.
In contrast, DR6, DRB1*0803 (subtype of
DR8), DR52, and DRB3*0301 were significantly less
frequent in patients with versus those without
anti-70K reactivity (Table 3). The DR6-associated
DRB 1* 1301 and DRB 1* 1302 and DRB 1*0803 alleles
were found exclusively in anti-70K-negative patients.
Two of 3 patients with the DR5-associated DRB1*1201
allele were anti-70K negative. These 4 DRBl alleles,
as well as DRB1*1102, share an amino acid sequence
Ile, Leu, Glu, Asp at positions 67-70 (16'LED7') in the
third hypervariable region of the DRBl p l domain.
This sequence was found in 6% of anti-70K-positive
patients, but in 69% of anti-70K-negative patients
(OR = 0.026, P = 0.00001) and 35% of normal controls
(OR = 0.11, P = 0.002). DR52 and one of its subtypes,
DRB3*0301, were also significantly less frequent in
anti-70K-positive patients. However, this negative
association was probably secondary to the lower frequency of the 167LED70sequence in some of the DR5-,
DR6-, or DR8-associated alleles, which are in linkage
disequilibrium with DR52.
Next, we examined the association of anti-70K
reactivity with the combination of a basic amino acid
residue at position 13 (R13 or HI3) and the 16'LED7'
sequence in the DRBl p l domain (Table 4). All
anti-U 1 RNP-positive patients with a combination of
the R13 or H13 positive and the 167LED70negative
belonged to the anti-70K-positive group. In contrast,
patients with a combination of the RI3 or H13 negative
and the 167LED70positive were exclusively in the
anti-70K-negative group. These results suggest that,
40 1
MHC CLASS I1 GENE ASSOCIATIONS WITH ANTI-U1 RNP
Table 4. Frequencies of a basic amino acid residue at position 13 (R” or HI3) and the sequence Ile,
Leu, Glu, Asp at positions 67-70 (167LED70)in the DRBl pl domain in 49 Japanese rheumatic disease
patients with or without reactivity to the U1 RNP 70K protein and in 43 normal controls
No. (frequency) of patients with anti-U1
RNP antibody
RI3 or HI3
16’LED70
Anti-70K positive
(n = 36)
Anti-7OK negative
(n = 13)
Norrnal controls
(n = 43)
Positive
Positive
Negative
Negative
Positive
Negative
Positive
Negative
2 (0.06)
29 (0.81)*
0 (0)t
5 (0.14)
2 (0.1s)
0 (0)
7 (0.54)
4 (0.31)
7 (0.16)
21 (0.49)
8 (0.19)
7 (0.16)
-~
~~
* Odds ratio (OR) = 106, P < 0.00001 compared with anti-70K-negative patients, and OR. = 4.3, P =
0.007 compared with normal controls.
t OR = 0.012, P < O.oooO1 compared with anti-70K-negative patients, and OR
compared with normal controls.
in the DRBl p l domain, a basic amino acid, arginine
or histidine, at position 13, is positively associated and
the 167LED70sequence is negatively associated with
anti-70K reactivity in a mutually exclusive manner.
In comparing the 31 patients with and the 18
without anti-C reactivity (Table 5 ) , we observed that
DR2, DRB 1*1502, and DQB 1*0601 were significantly
more frequent and DR8 was significantly less frequent
in anti-C-positive patients. The frequencies of DR2
(61%) and DRB 1* 1502 (55%) were also significantly
higher compared with normal controls (33% and 21%,
respectively; OR = 3.3, P = 0.02 and OR = 4.6, P =
0.003, respectively). The positive association with
DQB 1*0601is probably attributable to linkage disequilibrium with DRB1*1502, since DRB1*0803, which is
also in linkage disequilibrium with DQB1*0601, was
not associated with anti-C reactivity.
DR2 was found in 19 (79%) of 24 patients with
both anti-70K and anti-C reactivities, and in only 1
(4%) of the remaining 25 patients (OR = 91, P <
0.00001). Since DR2 was rarely found in patients with
either anti-70K or anti-C reactivity, these results suggest that DR2 simultaneously mediates antibody production against these 2 proteins.
There was no significant difference in HLA
frequencies between the 43 patients with and the 6
without anti-B/B’ reactivity. However, all 6 anti-BIB’negative patients had DR53 (DRB4*0101), versus 60%
of the anti-B/B’-positive patients (P = 0.08).
HLA associations with anti-U1 RNP antibody
titer. Anti-ENA antibody titers ranged from 1:160 to
r1:327,680 in patients with anti-U1 RNP antibody.
The anti-ENA titer was significantly higher in the 36
patients with anti-70K reactivity than in the 13 patients
without this reactivity (P = O.OOOl), but there was no
=
0.057, P = 0.007
relationship between anti-ENA titer and anti-A, B/B’,
or C reactivity. Therefore, patients with anti-U1 RNP
antibody were classified into 4 groups according to the
presence or absence of a basic amino acid residue,
arginine or histidine, at position 13 or the 167LED70
sequence in the DRBl p l domain, which were associated with anti-70K reactivity. The anti-ENA titer was
compared among these groups (Figure 1). The
anti-ENA titer was significantly higher in patients
negative for the 167LED70sequence than in those
positive for this sequence. Furthermore, in patients
without the 167LED70sequence, the anti-ENA titer
was significantly higher in those who had a basic
amino acid at position 13. It was noteworthy that all 8
patients having DRB 1*0401 demonstrated extremely
high anti-ENA titers (r1:163,840). HLA class I,
HLA-DQ, or DP associations with anti-ENA titer
were not identified.
DISCUSSION
In this study, we have demonstrated HLA class
I1 allele associations with anti-U1 RNP precipitin
antibody, as well as with irnmunoreactivities to the
70K and C proteins and antibody titer to U1 RNP.
These results can be summarized as follows: (a) anti-U1
RNP precipitin antibody was completely associated
with DQBl alleles possessing a combination of the
amino acid residues Y30 and T71RAELDT77;(b) this
antibody was also associated with unique DQB 1;
DRB 1 haplotypes; (c) anti-70K reactivity, which correlated with anti-ENA titer, was positively associated
with a basic amino acid residue, arginine or histidine,
at position 13 (DR2 or DR4) and negatively associated
with the 16’LED7’ sequence (some of DR5, DR6, and
KUWANA ET AL
DR8) in the DRBl p l domain; and (d) anti-C reactivity
was associated with DR2, especially with DRB 1* 1502.
These observations suggest that anti-U1 RNP autoantibody is associated with several shared epitopes
located on the hypervariable regions of HLA class I1
p l molecules. Recent structure analysis has revealed
the presence of the putative antigen-binding groove on
*em
z 327,680 -
a00
ee**o
0000
0
81,920 -
000
000
0
0
40,960 -
8888
00000
0
163,840-
20,480 -
Table 5. Frequencies of HLA-DRB1, DRB3, DRB4, and DQBl
genes in 49 Japanese rheumatic ,disease patients with anti-Ul RNP
antibody, according to the presence or absence of immunoreactivity
to the U1 RNP C protein
Phenotype
DRl
DR2
DR4
DR5
PR6
DR8
DR9
DR52
DRS3
DQ 1
DQ3
DQ4
Allele
DRB 1*0101
All combined
DRBl *I501
DRB 1* 1502
DRB 1* 1602
All combined
DRB 1*0401
DRB 1*0403
DRB 1*O405
DRB 1*0406
DRB 1*O407
DRB 1*0410
All combined
DRB 1* I101
DRB 1* 1102
DRB 1* 1201
All combined
DRB I * 1301
DRBl *I302
DRBl * 1305
DRB 1* 1401
All combined
DRB 1*0802
DRB 1*0803
DRB1*0901
All combined
DRB3*0101
DRB3*0202
DRB3*0301
DRB4*O101
DQB 1*0501
DQB 1*0502
DQB 1*0503
DQB1*0601
DQB 1*0602
DQB 1*0604
DQB 1*0301
DQB 1*0302
DQB 1*0303
DQB1*0401
DQB 1*0402
Anti-C positive
(n = 31)
no. Frequency
!i
19
;!
0.16
0.61*
0.06
17
2
1I:
0.55t
:I
1.
6
0
I.
0
4
I
0
21
21
1
1
1
0
41
?i
1
9
10
1
4.
2:
18
5;
3,
0
18:
2:
1
4.
11
9'
5
1
0.06
0.35
0.10
0.03
0.19
0
0.03
0
0.13
0.03
0
0.10
0.10
0.03
0.03
0.03
0
0.13$
0.10
0.03
0.29
0.32
0.03
0.13
0.06
0.58
0.16
0.10
0
0.58§
0.06
0.03
0.13
0.35
0.29
0.16
0.03
Anti-C negative
(n = 18)
no.
§
OR
=
4.8, P
=
0.02.
0.11
0.06
0
0.06
0
0.44
0.28
0
0.22
0.06
0
0.06
0.06
0
0.06
0
1
0
1
0
8
5
0
4
1
0
1
1
0
1
0
3
0
0.17
0
0.06
0.06
0.06
0.39
0.22
0.17
0.39
0.61
0
0.11
0.11
0.78
0.11
0
0.06
0.22
0
0.06
0.06
0.56
0.44
0.22
0.06
1
1
1
7
4
3
7
11
0
2
2
14
2
0
1
4
0
1
1
10
8
4
1
* Odds ratio (OR) = 27, 'f = ~.(1"1 (corrected P [~,,]
t OR = 21, P = O.ooOo5 (P,,,,=: 0.002).
t OR = 0.23, P = 0.04.
10,240 -
0
=
0.001).
0
5,1202,560-
000
c 1,280'
P
13R
0
0
Frequency
2
00
00
0r13H
e71LEmD
I
1
0.003
n
Positive
I Negative I
Positive
Negative
I
Negative
I
Positive
P = 0.0001
I
DRB1'0401
<
Positive
Negative 0
1
Figure 1. Titer of anti-extractable nuclear antigen (anti-ENA) antibodies in sera from 49 Japanese rheumatic disease patients with or
without a basic amino acid residue, arginine or histidine, at position
13 ("R or I3H) and the amino acid sequence isoleucine, leucine,
glutamic acid, aspartic acid at positions 67-70 (671LE70D)in the
DRBl PI domain. The anti-ENA titer was also compared according
to the presence or absence of DRB1*0401. Anti-ENA titer was
determined by the hemagglutination assay and the HLA class I1
alleles were determined by polymerase chain reaction restriction
fragment length polymorphism (see Patients and Methods). P values
were calculated by Wilcoxon rank sum test.
the surface of the MHC molecule, and the nature of
the amino acid residues in the groove is believed to
influence the overall shape of the groove, to control
peptide-binding, and to regulate immune response to
antigen (40,41). Therefore, it is suggested that shared
epitopes on HLA molecules mediate the binding of
processed autoantigens to the groove and regulate the
immune response, consisting in part of anti-U1 RNP
autoantibody production.
In this studv. we did not evaluate rheumatic
disease patients without serum anti-U 1 RNP antibody
as a control group, because anti-U1 RNP antibody is
not disease-specific (42). However, we believe that the
HLA class I1 alleles that were shown to be associated
with anti-Ul RNP antibody in our study are associ-
MHC CLASS 11 GENE ASSOCIATIONS WITH ANTI-U1 RNP
ated with the anti-UI RNP antibody itself, and not
with a specific type of rheumatic disease, since frequencies of DQB 1*0302 and DQB 1*0302;DRB1*0401
haplotype were not statistically different between
anti-U 1 RNP-positive patients who satisfied criteria
for SLE, SSc, or polymyositis/dermatomyositisand
those who did not.
There are 2 published papers reporting HLA
class I1 associations with anti-U1 RNP antibody by
genotyping analysis. First, Olsen et a1 (20) found a
positive association with DQB 1*0302 in North American Caucasian SLE patients with anti-U1 RNP antibody (compared with race-matched healthy controls),
which is consistent with our results in Japanese patients. Those authors also described greater frequencies, although not significantly greater, of DQB 1*0602
and DQB1*0301 in North American black patients
with SLE versus healthy controls, although they noted
a higher frequency of DQB 1*0501 compared with SLE
patients without anti-U 1 RNP and anti-Sm antibodies.
Another study of Japanese MCTD patients showed an
association of MCTD with DRB1*0401 and the
DQA1*03;DQB1*0301 haplotype (21). Since these 3
DQBl alleles (*0602, *0301, and *0302) possess the
amino acid sequence Y30 and T71RAELDT77, our
results partly support the idea that HLA-DQB1 gene
associations with anti-U1 RNP antibody appear consistent over many racial groups.
Serologically defined DR4 was reported to be
associated with anti-U1 RNP antibody in Caucasians
from various origins (12-16), but we did not detect a
DR4 association in Japanese, as described in a previous serologic analysis (17). If an ANA and HLA
association is postulated to exist worldwide among
many ethnic groups, this discrepancy could be explained by a different distribution of HLA class I1
alleles. In our results, anti-U1 RNP antibody was not
associated with the entire DR4, but with 1 specific
suballele of DR4, DRB1*0401, particularly with the
DQBl*0302;DRB 1*0401 haplotype. This haplotype is
shown to be one of the most frequent DR4-associated
haplotypes in many Caucasian ethnic groups, whereas
it is quite rare in Japanese (39). On the other hand, the
DQB 1*0601;DRB1* 1502 and DQB 1“0303;DRB1*0901
haplotypes, which are also associated with anti-U1
RNP antibody in this study, are major haplotypes in
Japanese, but are rarely detected in Caucasians (39).
Therefore, it is possible that the HLA association with
anti-U1 RNP antibody depends on the variable distribution of regional HLA class I1 alleles. We found that
anti-U1 RNP antibody was more frequently detected
403
in Japanese SSc patients than in North American
Caucasian patients (43), and further racial differences
in ANA distribution have been also noted in patients
with SSc (4345) and SLE (416). The precise reason for
these differences remains unclear, but the variation of
ANA frequencies among ethmic groups could similarly
be explained by the different distribution of HLA class
I1 alleles.
There are several replorts assessing HLA associations with immunoreactivity to individual U 1 RNP
constituent proteins. Hoffman and colleagues (47) first
noted that serologically determined DR4 and DR53
correlated with anti-70K reactivity in North American
Caucasians. Their further investigations employing
DNA typing showed correlations with the DR2- and
DR4-associated alleles (48), and DPB 1*0401 (49).
They also confirmed the association with DR2 and
DR4 in children (50). Our results support their findings, except with respect to DPB1*0401, which was
found in only 1 of our anti-70K-positive patients, but
in 3 anti-70K-negative patients and 4 normal controls.
The increased frequency of DPB1*0401 may reflect
its linkage disequilibrium with DRB 1* 1501 and
DRB 1*0401 in the North American Caucasian population (39). In addition, the negative trends toward DR5
and DR6 observed in their studies (47,48) are consistent with our finding of a “protective” effect of the
167LED70 sequence on anti-70K reactivity. We
showed, in addition, a new amociation between anti-C
reactivity and DR2, especially DRB1*1502. Although
no significant immunogenetic associations with anti-A
or anti-B/B’ reactivity were obtained, probably because of the small number of patients without these
reactivities, immunoreactivities to individual U 1 RNP
constituent proteins seem to be controlled by distinct
HLA class I1 genes.
Based on results of HLA genotyping analyses
of ANAs, Arnett and Reveille have speculated that
HLA-DQ associations are more primary than
HLA-DR associations in production of ANAs, including anti-U1 RNP antibody (51). However, all T cell
clones reactive with the U1 RNP particle generated
from the peripheral blood of anti-U1 RNP antibodypositive patients and healthy donors were shown to be
HLA-DR restricted (52), clearly indicating that the
HLA-DR molecule is involved in ANA production. In
the present study, we found HLA-DRB1 and DQBl
associations with anti-U 1 R.NP antibody production.
We believe that both the HLA-DR and DQ genes
control the anti-U1 RNP autoantibody response for
the following reasons: (a) anti-U1 RNP precipitin
404
KUWANA ET AL
antibody was completely associated with the shared
epitopes present in the 13QB1 Pl domain, whereas
anti-70K and anti-C reactivities and antibody titer
were well correlated with the HLA-DR genes; and (b)
anti-U I RNP antibody was associated with uncommon DQB 1;DRB1 haploi.ype, which cannot be explained by linkage disequdibrium alone, although it is
also possible that non-MH C genes in linkage disequilibrium with these haplotypes may contribute to this
antibody response. Taken together with our previous
observations on anti-DNA topoisomerase I antibody
response (4),it may be argued that ANA production is
not regulated by a single IILA-DR or DQ gene, but is
controlled by a combination of both genes. We should
note that dual control by HLA-DR and DQ molecules
has been observed in the immune response against
foreign antigens such as Schistosoma juponicum (53),
streptococcal cell wall antigen (54,551, and Cryptorneria japonica (Japanese cedar) pollen antigen (56).
Recent studies of ~immunogeneticassociations
with ANA have provided evidence supporting the
hypothesis that MHC class I1 molecules play a central
role in ANA production. This insight may provide
important clues to clarify the mechanisms of ANA
production and the path'ogenesis of systemic rheumatic diseases.
ACKNOWLEDGMENTS
We express our thanks to Tadayuki Sat0 and Kaoru
Sat0 (Tokai University Sch'ool of Medicine) for technical
assistance in serologic HL.A typing, and to Thomas A.
Medsger, Jr., MD (University of Pittsburgh School of Medicine) for his critical reading of the manuscript.
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class, major, histocompatibility, complex, small, antibody, immunoreactivity, relationships, antiu1, nuclear, ribonucleoproteins, associations, constituents, protein, genes, individual
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