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Contrasting molecular patterns of mhc class ii alleles associated with the anti-sm and anti-rnp precipitin autoantibodies in systemic lupus erythematosus.

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94
CONTRASTING MOLECULAR PATTERNS OF MHC
CLASS I1 ALLELES ASSOCIATED WITH THE ANTI-Sm
AND ANTI-RNP PRECIPITIN AUTOANTIBODIES IN
SYSTEMIC LUPUS ERYTHEMATOSUS
MARY L. OLSEN, FRANK C. ARNETT, and JOHN D. REVEILLE
Objective. To find evidence of a potential genetic
predisposition to the anti-Sm or anti-RNP precipitin
autoantibody responses.
Methods. HLA-DR and DQ alleles determined by
restriction fragment length polymorphism andor oligotyping in 49 subjects with either anti-Sm alone or
anti-RNP alone were compared with those in 139 racematched normal control subjects and 59 race-matched
lupus patients without anti-Sm and anti-RNP autoantibodies.
Results. Black patients with anti-Sm precipitin
had increased frequencies of HLA-DR2 and the DQw6associated DQAl*0102 (P = 0.007, odds ratio [OR] =
6.7) and DQB1*0602 (P = 0.001, OR = 9.1) chain
alleles compared with normal black control subjects.
Black patients with anti-RNP precipitin showed significant increases in the DQwS-associated DQA1*0101 (P=
0.03, OR = 5.5) and DQB1*0501 (P = 0.002, OR =
23.3) chain alleles compared with lupus patients without
anti8m or RNP. White patients with anti-RNP precipitin showed an increased frequency of the DQw8associated allele DQB1*0302 (P = 0.02, OR = 3.7)
-____
From the Division of Rheumatology and Clinical Immunogenetics, Department of Internal Medicine, The University of Texas
Medical School at Houston.
Supported by NIH grant AR-39325, an Arthritis Foundation
Clinical Research Center grant, and a grant from the Texas Gulf
Coast Chapter of the Arthritis Foundation. Dr. Reveille is an
Arthritis Foundation Arthritis Investigator.
Mary L. Olsen, MD: Division of Rheumatology and Clinical
Immunogenetics, Department of Internal Medicine; Frank C. Arnett, MD: Division of Rheumatology and Clinical Immunogenetics,
Department of Internal Medicine; John D. Reveille, MD: Division of
Rheumatology and Clinical Immunogenetics, Department of Internal Medicine.
Submitted for publication December 17, 1991; accepted in
revised form September 3, 1992.
Arthritis and Rheumatism, Vol. 36, No. 1 (January 1993)
compared with normal controls, as well as an increased
frequency of the DQwS-associated alleles DQA1*0101
and DQB1*0501 (P = 0.05, OR = 4.2) compared with
lupus patients without anti-Sm or RNP. There were no
specific HLA-DR2 or DR4 subtype associations found
with either anti-Sm or RNP precipitin autoantibodies.
Conclusion. There are distinct patterns of major
histocompatibility complex class I1 allele associations
with the anti-Sm versus the anti-RNP precipitin autoantibody responses, and HLA-DQ associations may be
more primary than HLA-DR associations.
Autoantibodies to the uridine-rich small nuclear
ribonucleoproteins (U snRNP), transcriptional enzymes for splicing messenger RNA, arise spontaneously in patients with the autoimmune disease systemic lupus erythematosus (SLE). In fact, anti-Sm
(Smith) autoantibodies, the autoepitopes of which
reside on U l , U2, U4, U5, and U6 snRNP, are highly
specific for SLE. Anti-RNP, which recognizes different antigens on U1 RNP, is often co-expressed with
anti-Sm in lupus patients or may occur alone in SLE or
in other systemic diseases, including mixed connective
tissue disease, scleroderma, and polymyositis (1).
Both anti-Sm and anti-RNP appear to occur more
commonly in blacks than whites with SLE (2).
SLE is a clinically and serologically heterogeneous disease in which genetic factors appear to play
an important role. Earlier studies have demonstrated
weak associations with the HLA-DR2 and DR3 alleles
in Caucasians (3,4). Stronger HLA class I1 correlations have been found for certain SLE autoantibody
subsets than for the disease itself (5-7). Such findings
suggest that the immune responses to these autoanti-
CLASS I1 ALLELES IN SLE
gens are genetically mediated by the major histocompatibility complex (MHC) and antigen-specific T
helper lymphocytes (8).
Prior studies assessing potential HLA phenotypic associations with the anti-Sm and anti-RNP
responses have been few in number, of limited sample
size, and often did not distinguish between patients
with anti-Sm and/or anti-RNP (6,9-18). Moreover, the
serologic HLA typing employed in these studies is
unable to detect the more extensive polymorphisms of
HLA class I1 alleles, which is now possible with
molecular methods. Bell and Maddison (15) and
Ahearn et a1 (6) found no HLA correlations with either
anti-Sm or anti-RNP, whereas Hamilton et al (12)
noted only a negative correlation with HLA-DQwl/
DQw2 heterozygosity. Anti-RNP has been correlated
with either HLA-DR4 (9-11) or the HLA-DQw3linked allele (18).
In a recent report by Hoffman et a1 (14), serologically determined HLA-DR4 and DRw53 were correlated with the presence of autoantibody specifically
to the U1 70-kd polypeptide snRNP antigen component of RNP. This same group of investigators has since
reported that a further analysis of 27 patients with
high-titer anti-U1 70-kd autoantibody showed an increased frequency of serologically determined HLADR2 and/or DR4, whereas, solid-phase direct DNA
sequencing implicated a shared amino acid epitope
within HLA-DRBl (16). Notably, the only study reporting any potential HLA association with the anti-Sm
response was that by Schur and coworkers (17). In
that study, HLA-DR7 was correlated with the presence of anti-Sm antibodies, which were found in 34%
of 106 Caucasian SLE patients studied. It is unclear if
those patients had concomitant anti-RNP antibodies.
Harley and coworkers reported the presence of increased HLA-DR4 with anti-Sm, but stated that there
was the concomitant presence of anti-RNP (13).
In the present study, 3 approaches were utilized
in an effort to characterize any HLA susceptibility
genes for the production of the anti-Sm or anti-RNP
autoantibody responses. Study subjects included both
black patients and Caucasian patients to allow assessment of genetic factors that cross racial barriers. HLA
frequencies were analyzed by autoantibody subsets
(anti-Sm alone and anti-RNP alone), since combining
all subjects with anti-Sm and/or anti-RNP might result
in too much heterogeneity, and thereby obscure a
potential HLA association. Immunogenetic differences were sought between these groups and racematched normal control subjects and race-matched
95
SLE patients who did not have anti-Sm or anti-RNP.
Finally, HLA alleles were identified via restriction
fragment length polymorphism (RFLP) and sequencespecific oligonucleotide (SSO) typing. These molecular genetic approaches afford more accurate identification of HLA class I1 alleles than do serologic
methods in diverse racial populations and allow identification of the actual (Y and p chain components (and
potential disease susceptibility sequence) of the individual HLA molecules.
PATIENTS AND METHODS
Study subjects. Forty-nine SLE patients (27 black
and 22 white) with either anti-Sm or RNP autoantibodies
were chosen for this study based on the concomitant availability of genomic DNA for HLA analysis by RFLP and
sequence-specific oligonucleotyping methods. Specifically
excluded from the analysis were any patients with both
anti-Sm and RNP precipitin antibodies. Comparison groups
were 59 race-matched SLE patients (36 black and 23 white)
without anti-Sm or RNP autoantibodies, and 139 racematched normal control subjects (61 black and 78 white)
with no personal history of autoimmune disease and whose
MHC alleles were similarly defined. Patients and controls
were drawn from the Houston (Texas) and Birmingham
(Alabama) areas and were proportionately matched for their
geographic origin.
Autoantibody analysis. Anti-Sm and anti-RNP autoantibodies in serum were detected by countercurrent immunoelectrophoresis using calf thymus extract as the antigen
source (19). In some patients, human spleen extract was also
used as antigen source to confirm the presence of anti-Sm.
RFLP analysis of HLA-DR and DQ alleles. All study
subjects underwent HLA-DR and DQ typing by standard
RFLP techniques. Genomic DNA was extracted from peripheral blood leukocytes (20), digested with the restriction
enzymes Tuq I and Barn HI, size-separated by overnight
electrophoresis through 0.8% agarose gels, transferred onto
nylon membranes (Zetabind; AMF-Cuno, Meriden, CT) by
Southern's method (21), and then hybridized to -yCTF'32labeled DRB 1 and DQB 1 complementary DNA probes and a
DQAl genomic probe as described previously (22-25).
HLA-DR and DQ specificities were assigned according to specific RFLP band patterns as described by the Tenth
International Histocompatibility Workshop (261, as well as
in our own (27,28) and others' studies (29-34). Individual
specific DQAl and DQBl chain alleles were assigned by
comparison with reported DQAl and DQBl RFLPs for
specific HLA-D haplotypes as determined by the WHO
(World Health Organization) Nomenclature Committee for
factors of the HLA system (35).
Sequence-specific oligonucleotide analysis of HLA-DQ
alleles and the HLA-DR2 and DR4 alleles. DQAl and DQBl
alleles were confirmed by oligonucleotide analysis of 42 of
the 49 patients with anti-Sm or anti-RNP. Twenty of the SLE
patients without anti-Sm or RNP autoantibodies and 40 of
the normal black controls were also analyzed by this addi-
OLSEN ET AL
96
0
DRBNIP-A
Mn0riO DRBl
5 '-CCCCACAoCLCOTTWTW
DRBANP-8
9 0 B O t i O DRBl
5
DRBAXP-2
DRBl Of DRZ
5 '-TmxmmwArnAAaAQQ
DRBAXP-4
DRBl Of DR4
5 '-0TTT~acAQOTTAMc
DRB3702
DRBl*lIOl, DRB1*1502,
DRB1.1601,
*-fxacmcA-
DRB1.1602
DRBl*llOl, DRBl*l502,
DYB5706
DRBL*lCOl, DRB1.1602
DRB7002
DRBl*l601
DRB7003
DRB1*1602
ORB7011
DRd1*1501, DRB1.1502,
DMI6Ol
DRB1+1502, DRBl*lSOl, DRBl*lCOZ
DILB.603
PBIO
DRBl*LSOl
t
DRB2813
DRB1.1503
DRB1.1503
DRZ group DRBL other t h m DRBl*1503
ORB3701
DRB1*0406
DRB3704
DR4 group DRBl othar than DRBI*OIOC
DRB5701
DR4 group DRBl othmr than DRB1*04OI
DRB5702
DRB7001
DRB1.0405
DR4 group DRBl other t h m DRB1.0401
or D R B ~ * O ~ O Z
DRB7005
DRB7006
DRB7007
DRBl*OlOl
DRB1.0403,
DRB1.0406,
DRBl*O407
DRB1*0402
Figure 1. Primers and probes used for HLA-DR2 and HLA-DR4 oligotyping. Except
for probe PS80 (see ref. 36), sequence information was obtained from circulars of the
Eleventh International Histocompatibility Workshop.
tional method. DNA was available for molecular subtyping
of the DRBI gene in 53 of the HLA-DR2 positive study
subjects and in 29 of the HLA-DR4 positive study subjects.
Genomic DNA underwent selective amplification by
the polymerase chain reaction (PCR) of the highly polymorphic gene segments of the second exon of HLA-DRB1,
DQAl, or DQBl using primers obtained from the Eleventh
International HLA Workshop (Figures 1 and 2). For each
PCR reaction, 1 pg of genomic DNA was mixed with PCR
buffer (50 mMTris HC1, pH 8.4,50 mM KC1, 1.5 m M MgCl,,
0.1 mdml gelatin); 0.2 mM each of deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine
triphosphate (dTTP); 25 pmoles each of the primers; 0.1 unit
of Ta9 DNA polymerase; and twice-distilled water into a
0.5-ml Eppendorf tube, to a total volume of 50 pl. Each
sample was then topped with 50 pl of mineral oil to prevent
condensation.
PCR amplification of the HLA-DQA1 region was
carried out at 95°C for 30 seconds, 55°C for 1 minute, and
72°C for 2 minutes, for 30 cycles. PCR amplification of the
HLA-DQB1 region was carried out at 90°C for 1 minute,
55°C for I minute, and 72°C for 2 minutes, for 30 cycles.
Finally, HLA-DR2 and DR4 were selectively amplified at
96°C for 30 seconds, 6 0 T for 30 seconds, and 72°C for 2
minutes, for 30 cycles.
SSO dot-blot hybridization was carried out employing multiple Y2P-ATP-labeled 18-mer probes corresponding
to nucleotide sequence polymorphisms in the outermost
domains of DQAl, DQBl, and DRBl (Figures 1 and 2). For
each dot-blot hybridization sample, 5 pl of sequence-specific
PCR product was mixed with 50 pl of 0.4 N NaOH and 25
mM EDTA, transferred to a sample well of the dot-blot
manifold containing a nylon filter prewet with distilled water,
vacuum pulled through the filter, and then washed with 100
CLASS I1 ALLELES IN SLE
Probes
97
s u e d Iicitv
DQA2501
DQAZ502
DQA1*0103, DQAlCOZOl, DQA2.0601
5 '-TWCAQTTCACCCATQA
DQAZ503
DQAl*0301, DQA1*0302
5 '-TQGQCAQTACAGCCATQA
DQA34Ol
DQAl.0101
5
DQA3402
DQAl.0102,
DQA3403
DQA1*0401, DQAl*OSOl
5 '-GAGACQAQCAGTTCTACQ
DQA4102
DQAl*OlO3
5 '-ACCTGQAGAAQAMQAQA
DQA5501
DQAl~OlOl, DQAl.0102,
DQAISOZ
DQAl.020
DQA5503
DQA1*0301, DQA1.0302
5 '-TCCGCAGATTTAQAAGAT
WAS504
DQA1.0401,
DQAl*OSOl, DQAl*OIOl
5 '-TCAQAmTTTAGAmG
DQA6901
DQAl.0101,
DQAlrO102, DQA1.0103
5 '-ATQGCTGTWCAAMCAC
DQA6902
DQAl*0201, DQAl.0301,
DQA1*0103, DQAL*OSOl
DQAl*Ol03
5 '-TCAGCIUATTTQQAIMTT
s
1
DQA1.0302
'-QAGATQAGGAGTTCTACQ
5 '-OAGATQAQCAQTTCTACQ
'-TCCACAQACTTAQATTM
5 '-ATcQC~OTOCTAAMCAT
DQ16903
D Q A P 0501
5 '-ATCQCTQTCTAAMCAT
DQA6904
DQA1+0401, DQA1.0601
5 '-ATCQCTGTGA-CAC
DQA7502
DQAl*OPOl, DQAl*O401, DQAl*OSOl
5 '-CTTQM~ATCCTQATTAA
DQA7504
DQA1.0501
5 '-CTTQMCAQTCWATTAA
DQW.301
DQB1*0401
5 '-QACCGAQCTCQTQCWQQ
DQB2302
DQB1*0402, DQB1*0303
5 '-MCGGQACCGAGCGCGM
DQ82601
DQB1*0501, DQB1.0502,
DQBl*0503
5 '-CGGGQTQTQACCAQACAC
~'-CQTTATQTOACCAGATAC
DQB260Z
DQB1*0601, DQBl*030l
DQB2603
DQB1*0602, DQB1.0302,
DQD2604
DQB1.0603,
DQB2606
DQB1.0605
5 '-CGTCTTQTMCCAGATAC
DQB3701
DQB140501, DQBl*OSOZ. DQB1*0503
5 '-AGGAGTACGTQCQCTTCG
0483702
DQB1.0601
5
DQB4501
DQB1*0301
5 '-QACGTQGAGQTGTACCQQ
DQB4901
DQBl*OSOl
5'-QGTQTACCWGCAGTGAC
DQ85701
DQBl*QSOl, DQBl*0604, DQB1.0605
5 '-CCOGCCTGTTQCCGAQTA
DQ85702
DQB1.0502,
5 '-QCQGCCTAQCQCCQAGTA
DQ85703
DQB1*0503, DQB1*0601
5 '-QQCGQCCTQACGCCGAGT
DQB5704
DQBl*OCOZ, DQBl*0603, DQBl*OSOJ
5 '-W!QGCCTQATQCCQAGTA
DQB5705
DQBl402Ol
5 '-GQCTQCCTGCCGCCQAGT
DQB5706
DQB1.0301,
DQB5707
DQB.0302
5 '-GGCCQCCTQCCGCCGAGT
DQB570a
DQB1*0401e DQB1.0402
5 '-GCGGCTTGACQCCGAQTA
0487002
DQBl*O601
5 *-GACCCGAGCGQAGTTQGA
DQB7003
DQB1*0602, DQB1*0603
5 '4AGQQUACCCQQQCQGAQ
DQB7005
DQBl*0201
5 '-GMACGQQC!QQCQQTQQA
DQBlr0303
DQ81.0604
DQB1.0504
DQBl*0303
~'-CQTCTTGTQACCAGATAC
5 '-CGTCTTGTMCCAGACAC
'-AGQAGGACGTQCQCTTCQ
5 '-GGCCQCCTQACQCCGAQT
Figure 2. Primers and probes used for HLA-DQA and HLA-DQB oligotyping. Sequence information was obtained from circulars of the Eleventh International Histocompatibility Workshop.
OLSEN ET AL
98
pl of T E (10 mM Tris HC1, 1 mM EDTA, pH 8.0). The filter
was then dried to completion by baking at 80°C for 1 hour.
The membrane was hybridized with each $*P-ATPlabeled probe in 5x Denhardt’s solution ( I x Denhardt’s
solution = 0.02% polyvinylpyrrolidone, 0.02% Ficoll, and
0.02% bovine serum albumin), 100 p g h l heat-denatured
herring sperm DNA, and TMAC solution (50 mM Tris HC1,
pH 8.0, 3.OM tetramethylammonium chloride, 2 mM EDTA,
0.1% sodium dodecyl sulfate [SDS]). Hybridization was
carried out at 54°C in a water bath, with constant agitation,
for 2 hours. Membranes were then rinsed with constant
gentle agitation in 100 ml of 2x SSPE, 0.01% SDS at room
temperature to remove excess probe, followed by washing in
100 ml of TMAC solution for 10 minutes at room temperature, and then 2 final washes in 100 ml of TMAC solution for
10 minutes at 58°C. Following the final wash, the membranes
were exposed to Kodak X-mat AR film with an intensifying
screen, at room temperature for 1-5 hours. The presence of
specific SSO probe reactivity in individual subjects allowed
DQAl, DQB1, and the DR2- and DR4-related DRBl allele
assignment.
Data analysis. Results of HLA typing black and
Caucasian subjects were analyzed separately. Within each
racial group, HLA comparisons were performed among 4
subsets as follows: 1) patients with anti-Sm alone (n = 16)
(12 black, 4 white), 2) patients with anti-RNP alone (n = 33)
(15 black, 18 white), 3) SLE patients negative for anti-Sm or
anti-RNP (n = 59) (36 black, 23 white), 4) healthy controls (n
= 139) (61 black, 78 white).
Two-tailed Fisher’s exact test was used for comparisons between the subsets. P values were corrected for
multiple comparisons (i.e., the number of alleles tested at a
particular locus), and both corrected (P,,,,)and uncorrected
P values are presented. Relative risks were calculated as
odds ratios (OR).
RESULTS
Table 1 outlines the HLA-DR and DQ specificities of the 4 study subsets in the group of black
subjects. Black patients with anti-Sm demonstrated an
increased frequency of HLA-DR2 (75%) compared
with healthy black controls (23%) or black SLE patients without anti-Sm or RNP autoantibodies (33%).
More striking in anti-Sm positives, as compared with
all anti-Sm negative groups, was the increased frequency of HLA-DQw6 (92%), which is in linkage
disequilibrium with some HLA-DR2 and DRw6 haplotypes. The increased frequencies of HLA-DR2 and
DQw6 in black patients with anti-Sm remained statistically significant after correction for multiple comparisons only in comparison with healthy controls. In
black patients with anti-RNP, there was a trend toward an increased frequency of HLA-DQw5 (47%)
when compared with SLE patients negative for
Table 1. HLA-DR and DQ specificities determined by RFLP
andor oligotyping analysis in American blacks with anti-Sm alone
or anti-RNP alone compared with black SLE patients negative for
anti-Sm or RNP and with normal black control subjects*
% Sm/RNP- % patients % patients
% control
HLA locus
DRI
DR2( 15,I6)
DR3( 17.18)
DR4
DR5( I I , 12)
DR6( 13)
DRq14)
DR7
DR8
DR9
DRwlO
DRw52a
DRwS2b
DRw52c
DRw53
DQw I(DQw5)
DQw I(DQw6)
DQw2(DQw2. I)
DQw2(DQw2.2)
DQw3. I(DQw7)
DQw3.2(DQw8)
DQw3.3(DQw9)
DQw4
subjects
(n = 61)
20
23
25
10
30
20
18
31
13
0
5
41
46
10
33
36
42
15
30
34
8
5
16
negative
SLE
patients
(n = 36)
0
33
25
3
33
17
11
31
19
0
3
28
47
17
33
14
53
22
31
33
3
6
8
with
anti-RNP
alone
(n
=
15)
13
40
27
7
40
20
13
7
20
0
6
47
53
0
7
47$
47
13
5
47
0
0
13
with
anti-Sm
alone
(n = 12)
0
75t
25
0
42
8
0
25
8
8
0
17
50
8
33
8
928
25
33
25
0
0
8
* RFLP = restriction fragment length polymorphism; SLE = systemic lupus erythematosus.
t P = O.OOO9, corrected P (Pco,) = 0.01, odds ratio (OR) = 10.1,
versus healthy controls. P = 0.01, P,,, = 0. I I , OR = 6.0, versus
Sm/RNP-negative SLE patients.
$ P = 0.02, P,,,) 0.16, OR = 5.4, versus Sm/RNP-negative SLE
patients. P = 0.04, P,, = 0.32, OR = 9.6, versus patients with
anti-Sm alone.
8 P = 0.002, P,,, = 0.02, OR = 14.8, versus healthy controls. P =
0.01, P,,,, = 0.08, OR = 9.8, versus Sm/RNP-negative SLE
patients. P = 0.01, P,,, = 0.08, OR = 12.6, versus patients with
anti-RNP alone.
anti-Sm or RNP antibodies (14%) or patients with
anti-Sm alone (8%) (Table 1).
Among Caucasians, only 4 patients had antiSm; two of them possessed HLA-DR2 and DQw6
(Table 2). The Caucasian patients with anti-RNP
showed a trend toward increased frequencies of HLADR4 and DQw8 when compared with healthy controls,
as well as a significant decrease in the frequency of the
HLA-DQw6 allele when compared with healthy controls or SLE patients without anti-Sm or RNP
(Table 2).
Actual DQAl and DQBl allelic frequencies in
99
CLASS I1 ALLELES IN SLE
Table 2. HLA-DR and DQ specificities determined by RFLP
andor oligotyping analysis in Caucasians with anti-Sm alone or
anti-RNP alone compared with Caucasian SLE patients negative for
anti-Sm or RNP and with normal Caucasian control subjects*
% patients % patients
HLA locus
DRI
DR2( 15,16)
DR3( 17,18)
DR4
DR5(11,12)
DR6( 13)
DR6( 14)
DR7
DR8
DR9
DRwlO
DRw52a
DRw52b
DRw52c
DRw53
DQwl(DQw5)
DQwl(DQw6)
DQwf(DQw2.1)
DQw2(DQw2.2)
DQw3.I(DQw7)
DQw3.2(DQw8)
DQw3.3(DQw9)
DQw4
% Sm/RNP% control
negative
subjects SLE patients
(n = 78)
(n = 23)
27
28
19
29
10
27
12
19
7
5
0
40
21
10
47
32
54
20
15
32
18
8
5
13
43
39
30
17
17
0
17
6
0
0
48
17
0
48
17
37
40
17
18
12
0
13
with
anti-RNP
alone
(n = 18)
with
anti-Sm
alone
(n = 4)
33
22
17
56t
5
0
11
11
11
0
0
17
50
50
25
25
25
0
0
0
0
0
0
25
25
0
25
11
0
61
39
17$
17
6
28
448
5
11
50
50
25
0
50
25
0
0
* See Table 1 for explanations of abbreviations.
t P = 0.03, Pcom= 0.33, OR = 3, versus healthy controls.
$ P = 0.004, P,, = 0.03, OR = 0.17, versus healthy controls. P =
0.02, Pcom= 0.16, OR = 0.18, versus Sm/RNP-negative SLE
patients.
8 P = 0.02, P,,,, = 0.16, OR = 3.7, versus healthy controls.
the 4 black study subsets are shown in Table 3. Black
patients with anti-Sm had significantly increased frequencies of the DQAl*0102 and DQB1*0602 chains of
DQw6, compared with healthy controls or SLE patients without anti-Sm or RNP antibodies. Black patients with anti-RNP demonstrated a significant increase in the DQA1*0101 chain of DQw5 when
compared with patients with anti-Sm or SLE patients
without anti-Sm or RNP antibodies. Also seen was a
significant increase in the DQB1*0501 chain of DQwS
when compared with SLE patients without anti-Sm or
RNP (P = 0.002, OR = 23.3). Caucasian patients with
anti-RNP showed significant increases in the
DQAl*0101 and DQB1*0501 chain alleles of DQw5
when compared with SLE patients without anti-Sm or
RNP antibodies (Table 4). Also seen was an increased
frequency of the DQB1*0302 chain allele of DQw8
when compared with healthy white controls (Table 4).
The HLA-DQ a chain first-domain amino acid
sequence information shows that the DQAl*O102
chain of DQw6 (increased in anti-Sm positives) is
identical to the DQA1*0101 chain of DQwS (increased
in both black and white patients with anti-RNP alone)
except at position 34, where the DQA1*0102 allele
carries a neutral glutamine residue rather than the
negatively charged glutamic acid residue of the
DQA1*0101 chain (Figure 3).
The HLA-DQ p chain first-domain amino acid
sequences of the DQB1*0602 chain of DQw6 (increased in anti-Sm positives) shares common sequences with either the DQB1*0501 chain of DQwS or
the DQB 1*0302 chain of DQw8 (increased in anti-RNP
positives), except at amino acid positions 9 , 5 7 , and 87
Table 3. DQAl and DQBl allele frequencies in American blacks
with anti-Sm alone or anti-RNP alone compared with black SLE
patients negative for anti-Sm or RNP and with normal black control
subjects*
% SdRNP-
% patients
% patients
with
anti-RNP
alone
(n = 15)
with
anti-Sm
alone
(n = 12)
HLA locus
subjects
(n = 61)
negative
SLE
patients
(n = 36)
DQA1*0101
DQA 1*O 102
DQAl*0103
DQAl*O201
DQAl*0301
DQA1*0401
DQAl*O501
34
31
I5
33
11
23
41
8
42
25
31
3
14
47
33t
40
13
7
7
27
40
DQBl*OS01
DQB 1*0502
DQBI*0503
DQB1*0601
DQB 1*0602
DQB 1*0603
DQB 1*0604
DQB1*0201
DQB I*0301
DQB I *0302
DQB 1*0303
DQB 1*0401
DQB 1*0402
25
5
3
6
14
31
8
8
408
7
% control
5
8
5
18
15
7
41
33
8
3
0
16
50
33
3
6
0
8
0
0
40
7
0
20
47
0
0
0
13
0
7%
17
33
0
8
50
8
0
0
8
6711
8
8
50
25
0
0
0
8
* SLE = systemic lupus erythematosus; OR = odds ratio.
t P = 0.04, OR = 5.5, versus patients with anti-Sm alone. P = 0.03,
OR = 5 . 5 , versus Sm/RNP-negative SLE patients.
$ P = 0.007, OR = 6.7, versus healthy controls. P = 0.05, OR = 4.2,
versus Sm/RNP-negative SLE patients.
8 P = 0.002, OR = 23.3, versus Sm/RNP-negative SLE patients.
11 P = 0.001, OR = 9.1, versus healthy controls. P = 0.04, OR = 4.6,
versus Sm/RNP-negative SLE patients.
OLSEN ET AL
100
Table 4. DQAl and DQBl allele frequencies in Caucasians with
anti-Sm alone or anti-RNP alone compared with Caucasian SLE
patients negative for anti-Sm or RNP and with normal Caucasian
control subjects*
% SdRNP-
% control
subjects
HLA locus (n = 78)
% patients
% patients
with anti-RNP with anti-Sm
alone
alone
(n = 18)
(n = 4)
negative
SLE
patients
(n = 23)
DQA1*0101
DQA 1 *O 102
DQA 1 *O 103
DQAl*0201
DQAl*0301
DQAl*0401
DQAl*O501
31
32
21
18
35
4
37
13
43
17
17
30
13
52
39t
17
DQB1*0501
DQBl*O502
DQB 1 *0503
DQB 1*0601
DQB1*0602
DQB 1 *0603
DQB 1 *Of34
DQB1*0201
DQB1*0301
DQBl*O302
DQB 1 *0303
DQB1*0401
DQBl*0402
27
3
5
3
27
17
10
32
32
18
8
0
5
13
4
0
0
39
17
0
56
22
39t
0
5
0
50
50
0
0
25
0
50
O$
17
50
11
229
50
0
0
0
50
0
0
25
50
25
0
0
0
17
0
0
177
33
44#
5
0
22
0
0
13
11
* See Table 3 for explanations of abbreviations.
t P = 0.05, OR = 4.2, versus Sm/RNP-negative SLE patients.
P = 0.03, OR = 0.22, versus healthy controls.
5 P = 0.05, OR = 0.26, versus SdRNP-negative SLE patients.
ll P = 0.01, OR = 0.15, versus Sm/RNP-negative SLE patients.
# P = 0.02, OR = 3.7, versus healthy controls.
$
(Figure 4). The amino acid differences at positions 9
and 87 are conservative, since a neutral residue is
replaced with another neutral residue. By contrast,
amino acid position 57 encodes for a negativelycharged aspartic acid in the DQB1*0602 chain, as
compared with a neutral valine in the same position of
DQBl*0501 and a neutral alanine in DQB1*0302.
11
DR 1,DQdS ( D Q A l 0 101 )
+
DR2,D@,6(DQA1*0102)tt
20
30
These charge differences may have etiologic significance in the anti-Sm response because all 12 black
patients and 3 of the 4 white patients with anti-Sm had
at least one DQAl chain with a neutral glutamine at
position 34. Moreover, both DQAl chains carried this
position-34 neutral glutamine in 8 of the 12 black
patients with anti-Sm. Also, 11 of the 12 black patients
with anti-Sm and 3 of the 4 white patients with anti-Sm
had at least one DQBl with a negatively charged
aspartic acid at position 57.
Molecular subtyping of the HLA-DRB 1 gene in
HLA-DR2 and DR4 positive black study subjects and
in Caucasian study subjects failed to reveal any specific subtype association with either the anti-Sm or
anti-RNP response. Among blacks with HLA-DR2,
the DRB1*1503 allele predominated in all groups of
patients and controls (70-86%), while in whites,
DRB1*1501 occurred in the majority (75-100%). Similarly, among HLA-DR4 positive lupus patients with
and without anti-RNP antibodies, as well as in HLADR4 positive normal controls, DRB1*0401 occurred in
43-53%, DRB1*0404 in 3543%, and the other DR4
subtypes occurred sporadically. Moreover, it is unlikely that significant Caucasian gene admixture has
occurred in these black study subjects since the
DRB1*1503 subtype has previously been shown to be
the most frequent DR2-DRB1 allele in American
blacks from the southern United States, with or without SLE (37).
DISCUSSION
This study demonstrates that precipitin autoantibodies to different U snRNP are associated with
different MHC class I1 alleles. Anti-Sm is correlated
with certain HLA-DR2, DQw6 haplotypes in black
SLE patients. Anti-RNP (U1 RNP), in the absence of
40
50
60
70
80
C O V m ~ ~ Q P S O O Y R I E F W D E E ~ L E R K ~ A ~ W P E F S K F G G F D ~ A L ~ M A V KRY
~KH~LNIMI
-----------------------Q---------------------------------------.------
Figure 3. HLA-DQ a first-domain amino acid sequences on selected haplotypes associated with anti-Sm alone (bottom)
or with anti-RNP alone (top). The DQw6 @chainDQA1*0102 is identical to the a chain of DQw5 (DQAI*OlOl), except at
position 34 (*), where DQA1*0102 carries a neutral glutamine (Q) residue rather than the negatively charged glutamic acid
(E) residue of the DQA1*0101.
101
CLASS I1 ALLELES IN SLE
10
IIR.
~ D ~ ~ S I D Q B A * OI~+ O I RDSPEDFVYQ FKGLCYFMG
~,H~,IQ,,#,(~BI."~"~)"
-.---.--F-
30
20
---n-----.
40
50
60
TERVRGWIW
I Y W R C DSDVGV~IAV
~ ~ ~
TFQGRPVAEY
-----'---y
-------A.--
70
WSQKLVLEG
80
ARASVDRVCR HHYEVAYRGI
'(11
i.VnR
. . _ _-_
- - - - -_
D - - -_ _
__
_ _T.
- -_
e L - T_
--- _
- - -_
- - - F_
--- _
Figure 4. HLA-DQ p first-domain amino acid sequences on selected haplotypes associated with anti-Sm alone (bottom)
or anti-RNP alone (top and middle). The DQw6 /3 chain DQB1*0602 shares common sequences with either the /3 chains of
DQw5 (DQB1*0501) or DQw8 (DQB1*0302), except at amino acid positions 9,57, and 87 (*). The differences at positions
9 and 87 are conservative, since a neutral residue replaces another neutral residue. Position 57 encodes for a negatively
charged aspartic acid (D) in the DQw6-associated DQB1*0602 chain, as compared with the neutral valine (V) in
DQB1*0501 and the neutral alanine (A) in DQB1*0302.
In contrast to patients with anti-Sm or SLE
anti-Sm, is associated with the linked alleles HLApatients without anti-Sm or RNP antibodies, our black
DR4 and DQw8 in Caucasian subjects and HLApatients with anti-RNP alone demonstrated an inDQw5 in black subjects. Moreover, analysis of the
creased frequency of HLA-DQwS. Our Caucasian
actual HLA-DQ chain alleles demonstrated an inpatients with anti-RNP showed an increase in the
crease in the DQw5-associated DQA1*0101 and
linked alleles HLA-DR4 and DQw8. In other studies
DQBl*0501 alleles in both black and white subjects
of European and American Caucasian subjects with
with anti-RNP, along with an increased frequency of
anti-RNP alone, increases in HLA-DR4 have been
the HLA-DQwtbassociated DQB 1*0302 allele in Causimilarly reported (9-1 1,13).
casian subjects with anti-RNP.
Serologically determined HLA-DR4 and
The HLA-DR2 and DQw6 alleles frequently
DRw53 were initially reported to be associated with
occur together due to linkage disequilibrium. Therethe presence of autoantibody specifically against the
fore, the gene promoting the anti-Sm autoantibody
anti-U1 70-kd polypeptide snRNP antigen component
could be either the HLA-DR2 or DQw6 allele, or
as well (14). The increased frequency of HLA-DR4
another as-yet-unidentified linked gene. Noteworthy is
found in those studies (9-11,13,14) may simply reflect
the absence of any specific HLA-DR2 subtype assothe linkage disequilibrium of HLA-DR4 with HLAciation with the anti-Srn autoantibody. Meanwhile,
DQw8. Most other studies either have not reported
evidence for the potential importance of the HLAHLA-DQ frequencies, or because of serologic HLA
DQw6 allele is based on the preferential increase in the
typing methods, could assess only the broad-spectrum
specific DQAl*0102 and DQB1*0602 chains in the
HLA-DQw3 specificity rather than its component
anti-Sm positive subjects (Table 3). The HLAHLA-DQw7, DQw8, or DQw9 subtypes. As our study
DQA1*0102 and HLA-DQB1*0602 chains comprise
assessed the actual DQAl and DQBl chain allelic
the HLA-DQ molecule found on the HLAfrequencies, the HLA-DQw8-associated DQB 1*0302
DR2(DRw15),Dw2,DQw6 haplotype. The other
chain was identified as increased in the Caucasian
HLA-DR2-associated
DQw6 haplotype,
patients with anti-RNP.
DR2(DRwlS),Dwl2,DQw6 is comprised of the HLAIn a more recent analysis of HLA associations
DQA1*0103 and DQB1*0601 chains. The remaining
with the U170-kd peptide snRNP antigen in Caucasian
DQw6 alleles are associated with HLA-DRw6 and
subjects, in which an association with HLA-DR2
include DRw6(DRw13),Dw18,DQw6 (DQA1*0103;
DQB1*0603) and H L A - D R W ~ ( D R W ~ ~ ) , D ~ ~ ~and/or
, D QDR4
W ~ was reported, HLA-DQ subtype analysis
showed a trend toward the HLA-DQw&associated
(DQA 1*0102; DQB 1*06O4).
DQB1*0302 chain as well (16). Finally, our study
Analysis of the linear amino acid sequence of
identified an increase in the HLA-DQw5-associated
the 4 possible DQBl chain alleles of DQw6 failed to
DQA1*0101 and DQB1*0501 chains in both black and
indicate a single sequence of DQB 1*0602 which disCaucasian patients with anti-RNP. No specific HLAcriminates it uniquely from all of the other DQw6p
DR4 subtype was found in association with the antichains. Perhaps the conformational tertiary structure
RNP response. It is notable that both the anti-Sm and
it obtains in conjunction with the DQAl chain is a
anti-RNP autoantibodies show stronger associations
critical determinant.
102
with HLA-DQ rather than the DR-linked alleles, since
other autoantibodies in SLE patients, including antiRo/SS-A, anti-LdSS-B, anti-dsDNA, and the lupus
anticoagulant, have each been correlated with
HLA-DQ alleles (38-40).
The anti-Sm and anti-RNP precipitin responses
appear to be correlated with specific HLA-DQ alleles
that demonstrate amino acid charge differences at
discrete areas within the second hypervariable region.
It is intriguing to postulate that these charge differences may play an important role in the complex
process of allorecognition and subsequent antibody
formation. It may be noteworthy that genetic predisposition to other autoimmune diseases, such as pemphigus vulgaris and insulin-dependent diabetes mellitus, has been correlated with the presence or absence
of an aspartic acid at position 57 of the DQ p chain
(41,42). Also, polymorphic DQ a and DQ p chain
interaction have been shown to be of importance in
HLA class I1 determinants of allorecognition (43,44).
Genetic predisposition to the anti-Sm and antiRNP precipitin responses remains complex and not
completely understood. It appears from our data that
HLA-DQ associations may be more primary than
HLA-DR associations. It should be emphasized that
our study identified anti-Sm and anti-RNP only by
immunodiffusion methods. It is possible that we did
not detect anti-Sm in some of the anti-RNP positives
(45) and/or that autoantibodies reacting with specific
subunits of the Sm/RNP complex will show stronger
associations with specific MHC alleles. Future studies
employing anti-Sm and RNP autoantibody characterization by immunoblot methods (4648) will be needed
to address potential HLA associations with the discrete polypeptide antigen subunits. Moreover, additional studies will be required to determine any role for
charge differences in the HLA-DQB chain position 57
and HLA-DQA chain position 34 which may predispose to the anti-Sm and RNP autoantibody responses.
Finally, it should be noted that both anti-Sm
and anti-RNP responses occur more commonly in
black than in white patients with SLE (2,49), and racial
differences in the frequencies of other autoantibodies
also have been noted (50,51). The reason for these
features is not known. Although there may be a
genetic explanation related to race, it seems unlikely
that HLA is the sole cause, since the potentially
relevant HLA-DR and DQ alleles still occur frequently in each of these racial groups.
OLSEN ET AL
ACKNOWLEDGMENTS
The authors wish to thank Karen Whittington for
technical assistance and Margaret Dougherty for preparation
of the manuscript.
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