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HLA-D Region genes associated with autoantibody responses to histidyl-transfer RNA synthetase Jo-1 and other translation-related factors in myositis.

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1240
HLA-D REGION GENES ASSOCIATED WITH
AUTOANTIBODY RESPONSES TO
HISTIDYLTRANSFER RNA SYNTHETASE (Jo-1) AND
OTHER TRANSLATION-RELATED FACTORS
IN MYOSITIS
ROSE GOLDSTEIN, MADELEINE DUVIC, IRA N. TARGOFF, MORRIS REICHLIN,
ANGELA M. McMENEMY, JOHN D. REVEILLE, NORANNA B. WARNER,
MARILYN S. POLLACK, and FRANK C.ARNElT
From the Division of Rheumatology and Clinical Immunogenetics,
Department of Internal Medicine, and the Department of Dermatology
and Internal Medicine, University of Texas Health Science Center at
Houston, and the Department of Microbiology and Immunology,
Baylor College of Medicine, Houston, Texas; and the Department of
Medicine, University of Oklahoma Health Science Center, the ArthritisiImmunology Program, Oklahoma Medical Research Foundation,
and the Veterans Administration Medical Center, Oklahoma City,
Oklahoma.
Supported by the Lupus Foundation of America, the Texas
Gulf Coast Chapter of the Arthritis Foundation, an Arthritis Foundation Clinical Research Center grant, Veterans Administration
grant CA-40552, and NIH grants AR-36546, AR-32214, and AI27181.
Rose Goldstein, MD, CM, FRCPC: Fellow of the Medical
Research Council of Canada, Division of Rheumatology and Clinical
Immunogenetics, University of Texas Health Science Center at
Houston (currently Career Scientist, Ontario Ministry of Health,
Health Research Personnel Development Program, and Assistant
Professor, University of Ottawa, Ottawa, Ontario, Canada);
Madeleine Duvic, MD: Associate Professor of Dermatology and
Internal Medicine, University of Texas Health Science Center at
Houston; Ira N. Targoff, M D Assistant Professor of Medicine,
University of Oklahoma Health Science Center, and Research
Associate, VA Medical Center of Oklahoma City and Oklahoma
Medical Research Foundation; Moms Reichlin, M D Professor of
Medicine, University of Oklahoma Health Science Center and VA
Medical Center of Oklahoma City, and Chief, Combined Immunology Section, Oklahoma Medical Research Foundation; Angela M.
McMenemy, MD: Fellow, Division of Rheumatology and Clinical
Immunogenetics, University of Texas Health Science Center at
Houston; John D. Reveille, MD: Assistant Professor of Medicine,
Division of Rheumatology and Clinical Immunogenetics, University
of Texas Health Science Center at Houston; Noranna B. Warner,
MD, PhD: Associate Professor of Medicine, Division of Rheumatology and Clinical Immunogenetics, University of Texas Health
Science Center at Houston; Marilyn S. Pollack, PhD. Associate
Professor, Department of Microbiology and Immunology, Baylor
College of Medicine; Frank C. Arnett. M D Professor of Medicine.
Division of Rheumatologyand Clinical Immunogenetics, University of
Texas Health Science Center at Houston.
Address reprint requests to Frank C. Arnett. MD, Division
of Rheumatology and Clinical Immunogenetics, Po Box 20708,
University of Texas Health Science Center at Houston, Houston,
TX 77225.
Submitted for publication November 22, 1989; accepted in
revised form February 27, 1990.
Arthritis and Rheumatism, Vol. 33, No. 8 (August 1990)
Myositis has been associated with HLA-B8 and
DR3, especially in white patients with polymyositis and
serum anti-Jo-1 antibodies. Twenty-eight patients with
myositis and serum translation-related autoantibodies
anti-Jo-1, anti-PL-7, anti-PL-12, anti-KJ, and antiSRP were studied for HLA class I1 specificities by
Southern blotting with HLA-DRP, DQP, and D Q a
probes. The association of HLA-DR3 (DRwl7) with
anti-Jo-1 antibodies in white myositis patients was
confirmed (P = 0.003, relative risk 8.9). However,
HLA-DRw52 haplotypes, regardless of subtype, were
present in all of the white and black patients with serum
anti-Jo-1 and other translation-related autoantibodies.
Moreover, one anti-Jo-1 positive patient had HLADRw8, an HLA-DRw52 haplotype on which the DRp3
gene has been partially deleted. No HLA-DQ specificity
or allele was common to all patients. The HLA-DR3,
DRS, DRw6, and DRw8 haplotypes, which bear the
HLA-DRw52 specificity, share the most homology in the
DRPl first hypervariable region at amino acid positions
9-13. Thus, this DRPl region appears to be the most
likely candidate “epitope” for translation-related autoimmune responses in inflammatory myositis.
Inflammatory myositis, particularly polymyositis in adults and dermatomyositis in children, has been
associated with HLA-B8 and DR3 in white individuals
(1,2). In an earlier study using serologic HLA typing
(3), the specific subset of myositis patients with serum
anti-histidyl-transfer RNA (tRNA) synthetase (antiJo-1) autoantibodies (4) had an even higher frequency
of HLA-DR3 than myositis patients overall; all 11
patients with serum anti-Jo-1 antibodies had either
HLA-DR3 and/or HLA-DRw6. It was hypothesized
that this autoimmune response in myositis may be
HLA-D AND ANTI-Jo-1 IN MYOSITIS
mediated at or near the HLA-D region. Recently, the
genetic structure of the major histocompatibility complex (MHC) has been more clearly defined ( 5 ) . Several
HLA-DR and DQ genes, each showing variable polymorphism and considerable linkage disequilibrium,
could be candidates for mediating the anti-Jo-1 response (5). Moreover, additional autoantibodies directed against other aminoacyl-tRNA synthetases and
other factors involved in intracellular translation have
been discovered, which are equally specific for myositis (6).
The studies reported here were designed to
investigate further the association of HLA-DR and
DQ alleles, determined by restriction fragment length
polymorphism (RFLP), in a new series of patients with
serum anti-Jo-1 (4) and other translation-related autoantibodies (6) known to be highly specific for myositis.
It was hypothesized that a single HLA allele or polymorphism might be common to all patients, both white
and black, with serum anti-Jo-1 antibodies, and that
this should be discernible by examining their genetic
profiles, especially in those who are HLA-DR3 nega-
tive.
PATIENTS AND METHODS
Study population. Sixteen unrelated adult patients (10
white and 6 black) with myositis and serum anti-Jo-1, and 12
patients (3 white and 9 black) with myositis and autoantibodies to other translation-related factors, including antipL-12 (alanyl-tRNA synthetase and tRNA"'") (n = 7). antipL-7 (threonyl-tRNA synthetase) (n = I), anti-KJ (n = 2).
and anti-SRP (signal recognition particle) (n = 2) were
studied. In addition, 19 patients (12 white and 7 black) with
myositis who had no autoantibodies to translation factors
were similarly studied. Patients were seen in Houston,
Oklahoma City, Baltimore, and as part of an ongoing study
in southern Georgia (7). All patients, with 1 exception (see
below), met criteria for the diagnosis of myositis proposed
by Bohan and Peter (8). Patients with myositis in the setting
of overlap with another connective tissue disease met independent criteria for the primary disorder, either systemic
lupus erythematosus (9) or scleroderma (10). One patient
had interstitial lung disease, arthritis, and serum anti-PL-12
antibodies and, to date, has not developed myositis. HLADR and DQ gene frequencies defined by RFLP analyses in
white and black patients with translation-related autoantibodies were compared with RFLP-defined HLA-DR and
DQ frequencies in 72 white and 45 black controls. Controls
were normal race-matched volunteers, including medical
students and university and hospital personnel.
Autoantibody determinations. Serum samples from
all patients were tested by indirect immunofluorescence on
HEp2 cells and by Ouchterlony immunodiffusion and/or
countercurrent immunoelectrophoresisagainst concentrated
bovine tissue (liver, thymus, or spleen) extracts (1 1). Each
1241
serum sample that tested positive by immunofluorescenceor
immunodiffusion was tested further by protein A-assisted
immunoprecipitation by the method of Forman et a1 (12).
using unlabeled HeLa cells. All sera were also tested specifically for the myositis-associated translation-related antibodies (6). anti-Jo-1 (4,13,14), anti-KJ (15), anti-PL-7
(16,171, and anti-PL-12 (18). Solid-phase enzyme-linked
immunosorbent assays using immunoaflinity-purifiedantigen
were used to test for antibody to Jo-1 (14) and to KJ (15). an
as-yet-unidentifiedtranslation factor. Each serum was tested
for inhibition of threonyl-tRNA synthetase (PL-7) activity
(19) and alanyl-tRNA synthetase (PL-12) activity (18), using
a sensitive and specific method for detection of anti-PL-7
and anti-PL-12 antibodies, respectively. Anti-SRP antibodies
were detected by immunoprecipitation and confirmed by
immunoblotting (20).
HLA-DR and HLA-DQ analysis by Southern blotting.
Genomic DNA was extracted from peripheral white blood
cells, or in some cases, from Epstein-Barr virus-transformed
lymphoblasts, by a standard proteinase K digestion, followed
by phenol-chloroform extraction and ethanol precipitation
(21). DNA samples (10 pg) were digested with 5 units of the
appropriate restriction enzyme per microgram of DNA,
according to the manufacturer's instructions (Bethesda Research Laboratories, Gaithersburg, MD). The digested DNA
was electrophoresed in 0.8% agarose gels and transferred to
nylon membranes (Zetabind; Cuno, Meriden. CT) by the
method of Southern (22). Prehybridization and hybridization
were carried out, according to the manufacturer's instructions, at 42°C in the presence of 100 &ml of sheared
denatured herring sperm DNA. Each probe insert was
isolated using low-melting agarose and labeled with "PadCTP (Amersham, Arlington Heights, IL) by the random
priming method (Boeringer Mannheim, Indianapolis, IN)
DRw 52
Haplotype Taq I-DRB
HLA-DRw6
10 Kb
HLA-DRw8
-
-
~
2Barn4 HI-DOa24Kb
Dw26
Taq I-DQo 5.5Kb
~
Or
Dw26
undefined DRw52 allele
Figure 1. A scheme for HLA-DRw52 allelic subtyping by restriction fragment length polymorphism analysis. In HLA-DR3 haplotypes, the Tuq I DRB 10-kb fragment (DRHTuq I 9.60) defines the
Dw24 (DRw52a) allele, and in HLA-DR3 and HLA-DR5 haplotypes, the Taq 1 DRP 12-kb fragment (DRpITuq I 11.46)defines the
Dw25 (DRw52b) allele. In HLA-DRw6 haplotypes, the Tuq I DRp
12-kb fragment defines Dw25, and the Tag 1 DRB 10-kb fragment
defines either Dw24 or Dw26. The Dw26 (DRw52c) allele is further
identified by a Bum HI DQa 2 4 k b fragment (DQNBqm HI 23.93)
and a Taq I DQa 5.5-kb fragment (DQNTuq 1 5.50).
GOLDSTEIN ET AL
1242
1
9
2
Kb
-12
-10
4
S
O
-
Kb
I
24
-6.5
-
10
-0.2
A)
1.0 I-DFIB
B)Teq I-Wa
C) barn H C W a
Figure 2. Southern blot analysis of restriction fragment length
polymorphisms used for HLA-DRw52 subtyping. A, Taq I-digested
genomic DNA hybridized to the HLA-DRp probe, showing the
10-kb (lane 1) and 12-kb (lane 2) fragments (see Figure 1). B, Taq
I-digested genomic DNA hybridized to the HLA-DQa probe,
showing the absence (lane 3) and the presence (lane 4) of the 5.5-kb
fragment associated with Dw26. C, Barn HI-digested genomic DNA
hybridized to the HLA-DQa probe, showing the absence (lane 5 ) and
the presence (lane 6) of the 24-kb fragment associated with Dw26.
(23,24). After hybridization, membranes were washed to a
final stringency with 0.1x SSC ( l x SSC = 0.15M NaCI,
0.015M sodium citrate, pH 7.01, 0.1% sodium dodecyl sulfate at 65°C for 30 minutes, air dried, and exposed to Kodak
XAR-5 film for 1-7 days. The probes that were used were
complementary DNA probes for DQP (800-basepair Pst
IIEco RI fragment) (25) obtained from Dr. D. Larhammer
(Uppsala, Sweden), DRP (700-bp Psr I fragment) (26) obtained from Dr. E. Long (Bethesda, MD),and a genomic
probe for DQa (2.4-kb Pst I fragment) (27) obtained from Dr.
J. Trowsdale (London, UK).
Interpretation of restriction fragment patterns. The
Tuq I RFLP patterns seen with the DRP probe were used to
assign DR specificities DRl through DRw8, DRw52, and
DRw53, and the Taq I DQa,Barn HI DQa, and Barn HI DQB
RFLP patterns were used to assign DQ specificities, according to the findings of the Tenth International Histocompatibility Testing Workshop (28) and other studies (2P-31). The
HLA-DRw52 allelic subsets Dw24 (DRw52a). Dw25
(DRw52b) (28,32), and Dw26 (DRw52c)(28,33) were defined
by RFLP, as summarized in Figures 1 and 2 (34.35). Specific
Table 1. Restriction fragment length polymorphism-determined HLA class I1 specificities among
patients with myositis and with serum translation-related autoantibodies*
Antibody,
patienurace
Anti-Jo- I
l/W
2nv
3nv
4nv
5iw
6Mr
7nv
8nv
9nv
ow
1
1I/B
1YB
13lB
14/B
I51B
16/B
Anti-PL-7
17/B
Anti-PL- 12
18Mr
19/W
20lB
21lB
22lB
23lB
24lB
Anti-KJ
25Mr
26/B
Anti-SRP
27lB
28lB
Diagnosis
HLA-DR
HLA-DQ
2.1.7
2.1,8
2.1.7
2.1,6
2.1,6
2.1.6
2.1.6
6.6
2.1.7
7.7
2.2.4
PM, ILD
PM, ILD
PM. ILD
PM, ILD
PM, ILD
PM, ILD
PM, ILD
PM, ILD
PM, ILD
PM. ILD
PM, ILD
PM, ILD
PM, ILD
PM, ILD
PM, ILD
PM, ILD
3(~17),~6(~14),~24,~25
3(w 17),4,w24,53
3(w17),5(w I l),w24,w25
3(w 17). w6(w 13). w24, w25
3(w 17),2,w24
3(w17),2,w24
3(w1 7 ) , w 6 ( ~ 1 3 ) , ~ 2 4 , ~ 2 6
W6(W13),W24
5(wl1),4,w25,53
5(wll),w25
3(w18),7,w24,53
w8, w6(w 14),w24
w q w 13),w6(w14),w25,w26
S(wl1),7,w25,53
w8t.l
5(w 1l),w6(w13).w25,w26
PM, ILD
w6(w 13),w24,w26
DM, ILD
PM, ILD
A, ILD
DM, ILD
PM
PM, ILD
PM, ILD
5(wl1),2,w25
3(w17),w6(w13),w24
3(w 17),2,w25
w6(w14),2,w25
5(wl1),2,w25
5(11),7,w25,53
3(wl8), 1 0 . ~ 2 4
PM, ILD
PM, ILD
5(wl l),w8,w25
5(wl1),2,w25
2.1.6
5 97
PM
PM
5(wl1),2,5
5(wl1),7,w25,53
6,7
ND
* PM = polymyositis; ILD = interstitial lung disease; DM = dennatomyositis; A = arthritis, no
myositis; ND = not determined.
t Patient 15 was serologically positive for DRw52.
HLA-D AND ANTI-Jo-1 IN MYOSITIS
DQa and DQp chains, previously defined by 2-dimensional
gel electrophoresis, were assigned from DQa and DQP
RFLP patterns suggested by the Tenth International HistocompatibilityTesting Workshop (36). Finally, an HLA-DQP
Hinc I1 15-kb RFLP, previously shown to be associated with
a subset of HLA-DR3 and HLA-DR7 haplotypes and with
myasthenia gravis (37). was studied.
Statistical analysis. Comparisons between groups
were analyzed by the chi-square test with Yates' correction
or Fisher's exact test with 2-tailed probabilities (when appropriate). The Mantel-Haenszel chi-square test was used
when whites and blacks were analyzed together. P values
less than 0.05 were considered statistically significant. These
values were not corrected, since an association of serum
anti-Jo- 1 antibodies with HLA-DR3 has been reported
previously (3), and we were specifically seeking alleles
related or linked to HLA-DR3, especially in blacks and in
HLA-DR3 negative patients. The relative risk (RR) for a
given HLA-D specificity was determined as the odds ratio.
RESULTS
HLA-DR alleles, by RFLP typing, in myositis
patients with serum translation-related antibodies. The
HLA-DR and DQ specificities were determined by
RFLP analysis of 28 (13 white and 15 black) myositis
patients with serum translation-related autoantibodies
anti-Jo-1 (n = 16), anti-PL-7 (n = I), anti-PL-12 (n =
7), anti-KJ (n = 2), and anti-SRP (n = 2) (Table I).
Twenty-five (8%) of 28 patients with serum translation-related autoantibodies had interstitial lung disease
(Table I), a finding that is consistent with previous
reports (6,3WO).
HLA-DR3 (DRwl7) was again associated with
I243
serum anti-Jo-I antibodies in white myositis patients,
occurring in 7 of 10 patients (P = 0.003, RR 8.9)
(Tables 1 and 2). Of the DQ specificities, only the
frequency of HLA-DQw2.1, present in 8 (80%) of 10
white myositis patients with serum anti-Jo-1 antibodies, was significantly increased compared with white
controls (P = 0.0004, RR 15). Three white anti-Jo-l
antibody positive patients who were HLA-DR3 negative had other DRw52 haplotypes (2 with DR5 and I
with DRw6) (Table 1). In the small number (n = 6) of
black myositis patients with serum anti-Jo-1 antibodies, none of the individual DRPl or DQ specificities
were significantly different from those in black controls. All were DQw2.1 negative, and only 1 had DR3;
however, HLA-DRw52 bearing haplotypes were
found in all. The frequency of HLA-DRw52 was
significantly increased in white patients with serum
anti-Jo-1 antibodies compared with race-matched controls (P = 0.03, RR > 4 . 3 , but was not significantly
increased in the small number of black patients (Table
2). Patient 15 (Table 1) was particularly notable in that
she possessed HLA-DRw8 on 1 haplotype and DR1 on
the other. HLA-DR1 is not associated with DRw52;
however, DRw8 positive individuals, including this patient, were typed as DRw52 positive by serologic methods, but show only 1 DRP gene by molecular methods.
Recent data suggest that DRw8 is a fusion gene composed of portions of DRPl and DRm; its coding sequences show striking homology to those of the DRPI
alleles DR3, DR5, and DRw6, while its 3'-untranslated
region is identical to that of DRm (41).
Table 2. Frequencies of restriction fragment length polymorphism-determinedHLA class I1 specificities among white and black inyositis
patients with serum anti-Jo-l antibodies*
Specificity
DR3(w17)
DR3(w18)
DR4
DRXw 1 I )
DRw6(w13)
DRw6(w14)
DRw8
DRw52t
Dw2402a)
Dw25(52b)
Dw26(52c)
DRw53
DQw2.I
DQw2.2
White
patients
(n = 10)
White
controls
(n = 72)
7 (70)
0
2 (20)
3 (30)
3 (30)
1 (10)
0
15 (21)
0
21 (29)
10 (14)
20 (28)
10 (loo)
8 (80)
5 (50)
l(10)
2 (20)
8 (80)
7 (10)
5 (7)
48 (67)
29 (40)
15 (21)
7 (10)
34 (47)
I5 (21)
11 (15)
P
RR
0.003
NS
NS
NS
8.9
-
-
NS
NS
NS
0.03
0.04
NS
NS
NS
>4.5
5.9
O.OOO4
I5
-
-
-
Black
patients
(n = 6)
0
1(17)
0
2 (33)
2 (33)
2 (33)
2 (33)
6 (100)
2 (33)
3 (50)
2 (33)
2 (33)
0
Black
controls
(n = 45)
P
RR
5 (11)
NS
NS
7 (16)
13 (29)
8 (18)
8 (18)
4 (9)
36 (80)
20 (44)
21 (47)
4 (9)
14 (31)
5 (11)
13 (29)
NS
NS
NS
NS
NS
NS
NS
NS
NS
-
8 (18)
NS
NS
-
-
-
I(17)
NS
* Values are the number (%I of individuals with a given specificity. RR = relative risk; NS = not significant.
t p = 0.002, RR 4.1, for combined allelic frequencies of DRw52 in white and black anti-Jo-I positive patients compared with normal
race-matched controls, by Mantel-Haenszelanalysis. p = 0.01. RR 4.4, for combined allelic frequencies of DRw52 in white and black anti-Jo-1
positive patients compared with race-matched myositis patients without translation-related autoantibodies, by Mantel-Haenszel analysis.
0
NS
-
GOLDSTEIN ET AL
3 244
Table 3. Frequencies of the Hinc I1 15-kb HLA-DQP restriction fragment length polymorphism in
white and black HLA-DR3 andlor HLA-DR7+ myositis patients and controls*
HLA-DR
type
White patients
White controls
Black patients
Black controls
HLA-DR3 ,X
HLA-DR7, y
HLA-DR3.7
3/14 (21)
112 (50)
112 (50)
3/21 (14)
213 (67)
515 (100)
619 (67)
212 (100)
0
416 (67)
313 (100)
0
* x = an HLA-DR antigen that is not DR7; y = an HLA-DR antigen that is not DR3. No significant
frequency differences between patient and control groups of either race were observed. Values are the
number &).
DRw52 gene frequencies in 10 white anti-Jo-1
positive patients were significantly increased compared with the DRw52 frequency in 12 white myositis
patients who had no detectable serum translationrelated antibody (16 of 20 [80%]versus 11 of 24 [46%];
P = 0.04, RR 4.7). The combined DRw52 allelic
frequencies in white and black patients with serum
anti-Jo-1 antibody were highly significantly different
than those of race-matched controls (P = 0.002, RR
4.1) and those of 12 white and 7 black myositis patients
with no detectable serum translation-related antibodies (P = 0.01, RR 4.4), when analyzed by the MantelHaenszel chi-square test.
Similar to the anti-Jo-1 positive patients, all
white and black patients with any other translationrelated autoantibody possessed DRw52 haplotypes
(Table 1). When all 28 myositis patients with serum
translation-related autoantibodies (anti-Jo- 1, anti-PL7, anti-PL-12, anti-KJ, and anti-SRP) were analyzed,
the association with the HLA-DRw52 phenotype was
significant in white patients compared with racematched controls (P= 0.02, RR > 6). The difference
between black patients and black controls was not
significant (P = 0.1, RR > 3.5). The DRw52 allelic
frequency was also higher than control frequencies,
among white patients, but not among black patients,
with translation-related antibodies (whites P = 0.0003,
RR 5.8; blacks P = 0.5, RR 1.4). When analyzed
together (by Mantel-Haenszel chi-square test), the
DRw52 phenotypic and allelic frequencies in whites
and blacks with serum translation-related antibodies
were both higher than in race-matched controls (phenotypic frequency P = 0.05, RR 4.7; allelic frequency
P = 0.003, RR 2.7).
The Dw24 (DRw52a) subtype defined by RFLP
analysis was significantly increased in white myositis
patients with anti-Jo- 1 antibodies compared with
white controls, occurring in 8 (80%) of 10 patients ( P =
0.04, RR 5.9) (Table 2). This is probably attributable to
the presence of HLA-BS and DR3 in 7 of these 8
patients, as the HLA-BS, DR3 haplotype is known to
always carry the HLA-Dw24 (DRwS2a) allele (33). No
DRwS2 subtype appeared to be increased in the black
myositis patients with serum anti-Jo-1 antibodies.
Among the 12 myositis patients (3 white and 9 black)
with the non-anti-Jo- 1 serum translation-related autoantibodies (patients 17-28, Table 11, 9 (75%) had
HLA-Dw25 (DRw52b), which was significantly
greater than the frequency in race-matched controls (P
= 0.04, RR 5 , by Mantel-Haenszel chi-square test).
HLA-DQ alleles, by RFLP typing, in myositis
patients with serum translation-related antibodies. No
HLA-DQ alleles were common to all of the serum
anti-Jo- I positive patients. Notably, HLA-DQw2.1,
which was significantly increased in whites with antiJo-1 antibody by virtue of its linkage to HLA-DR3
(DRwl7), occurred in none of the blacks who were
anti-Jo-1 positive. In addition, no DQa or DQP chain
genes determined by RFLP were common to all of
these patients. In a random subset of both white and
black myositis patients with HLA-DR3 and/or HLADR7, there was no increase in frequency of the H i m I1
15-kb DQp fragment compared with race-matched and
DR3- and/or DR7-matched controls (Table 3).
DISCUSSION
In this study, the previous association of serologically defined HLA-DR3 with the anti-Jo-1 response in whites (3) was confirmed, and the HLADw24 (DRw52a) and DQw2.1 specificities, which are
in linkage disequilibrium with the white HLA-BS,
DR3 (DRw 17) haplotype, were also significantly increased in patients with this autoimmune response.
Nonetheless, 3 whites with serum anti-Jo-1 antibody
were negative for DR3 (DRwl7) and DQw2.1, and 2
were negative for Dw24 (DRw52a). Moreover, among
6 American blacks with serum anti-Jo-1 antibody,
only 1 possessed HLA-DR3 (the DRw18 subtype
associated with DQw4), none had DQw2.1, and only 2
had Dw24 (DRw52a); no DQa or DQP chain gene was
common to all of these patients. Although the number
of black patients with serum anti-Jo-1 antibody was
small, the only HLA specificity found in all patients of
both races with serum anti-Jo-1 antibody was the
HLA-D AND ANTI-.To-1 IN MYOSITIS
supertypic DRw52, which was not restricted to any of
its recognized subtypes (Dw24 [DRw52a], Dw25
[DRw52b], Dw26 [DRw52c], or the even less welldefined DRw52 occurring with DRw8 [5,33]). Moreover, HLA-DRw52 (predominantly the Dw25
[DRw52b] subtype) was also found in all 12 patients
with other autoantibodies directed against translation
factors (PL-7,PL-12, KJ, and SRP). Thus, DRw52
appears to be the strongest HLA-DR or DQ marker
for myositis associated with autoantibodies to Jo-1 and
other translation-related factors. Other loci on these
haplotypes, particularly on the HLA-A1, B8, DR3
haplotype in whites with Jo-1, may also be important
in the pathogenesis of these immune responses.
Since HLA-DRw52 occurred in all the myositis
patients with serum antibodies against translationrelated factors of both races, and no HLA-DQ alleles
were common to all, it appears likely that HLA-DR
genes rather than HLA-DQ genes are involved in
these autoimmune responses. The molecular genetic
basis for the DRw52 serologic specificity that is coexpressed with DR3, DR5, DRw6, and DRw8 is not fully
understood (32-35,42-44). Sequence homologies
among DR3, DR5, and DRw6, as well as the DRw52
subtypes (Dw24-Dw26), suggest that DRwSZbearing
haplotypes constitute a “gene family” that is evolutionarily distinct from other haplotypes (42,44,45)
(Figure 3). The HLA-DRw8 haplotype appears to
have evolved from the DRw52 family, but because of
a large deletion encompassing the 3‘ untranslated
portion of DRPl through the coding sequences of
01
P2
1245
Table 4. Amino acid sequences at positions 9-13 of the HLADRPl chains of the DRw52 haplotypes*
Amino acid position
DR3
DR5
DRw6
DRw8
9
10
II
12
13
Reference
Glu
Tyr
Ser
Thr
-
Ser
-
29, 54
55
29, 54
54
-
-
-
-
-
-
Gly
* - denotes identity with DR3pI residue at that position.
DRP3 (411, it bears only 1 DRP gene (42,431. The
resulting fusion gene is composed of the DRPl coding
sequences that show homology with HLA-DR3, DR5,
and DRw6, and the 3’ untranslated portion of the
DRP3 gene.
This observation may be relevant to the association of DRw52 with myositis autoantibodies in this
study. Patient 15 (Table 1) had the typical HLA-DRw8
and DRI RFLP and DRw52 serologic specificity (46).
and therefore, should have only DRPI, and no DRP3,
coding sequences. Thus, it is possible that DRw52 and
the myositis-associated “epitope” are encoded from
DRPl alleles. Nonetheless, the DRP3 alleles and/or
DRw52 serologic specificity remain alternative candidates for underlying susceptibility to the translationrelated immune response.
The DRPl -encoded first hypervariable region
amino acid positions 9-1 3 (Glu-Tyr-Ser-Thr-Ser) are
shared by DR3. DR5. and DRw6, with DRw8 differing
only at position 13 (32,47,48) (Table 4). There is less
@3
P4
a
DRw8
OR2
DR 1
Figure 3. Schematic representationof DR genes of different haplotype families. The number of DRP
genes varies on different haplotypes. + = expressed DRPl sequences; I/I = pseudogene; w53 =
expressed D R P sequences; a = expressed DRa sequences; w52 = expressed DRp3 sequences.
(Reproduced. with permission. from Arnett FC. Bias WB. Reveille JD: Genetic studies in SLE and
Sjogren’s syndrome. J Airtoimmun 2:403413. 1989.)
GOLDSTEIN ET AL
1246
homology among these same DRPl molecules in the
second and third hypervariable regions, and no other
DRP sequences show similar homology in this first
hypervariable region. Also, in the first and third hypervariable regions encoded by the DRP3 alleles,
Dw24 (DRw52a) differs from Dw25 (DRw52b) and
Dw26 (DRw52c) only at amino acid positions 1 1 and
74, respectively (33). HLA-DR3 DRPI, but not DRS,
DRw6, or DRw8, shares a common third hypervariable sequence with Dw24 (DRw52a) by virtue of a
reported gene conversion event (32). Thus, the amino
acid sequence shared by DR3. DR5, DRw6, and DRw8
(DRPl amino acid positions 9-13) seems t o us to be the
most likely candidate as an "epitope" promoting the
Jo- 1 and other translation-related autoantibodies in
myositis. Transposition of this sequence onto the
postulated 3-dimensional structure of the class I1
MHC molecule would map it to the floor of the
peptide-binding groove (49-5 I), where it thus might be
available for a physical association with as-yetundefined antigen(s) promoting these autoimmune responses. Interestingly, this same gene region has been
implicated recently in human immune responsiveness
to a ragweed antigen (Rye 111) (52).
The autoantibodies to the aminoacyl-tRNA
synthetases are found almost exclusively in the serum
of myositis patients and react selectively with only the
I specific synthetase targeted by the autoantibodies in
each patient's serum. This selectivity suggests that
production of these autoantibodies is antigen driven
(18). A recent report by Bunn and Mathews (53)
defines the binding site of anti-PL-12 sera for tRNA"'"
as the 7-9 nucleotide sequence of tRNA"'" that contains the unique anticodon loop, supporting the importance of the specificity of these autoantibodies. However, anti-PL-12 sera have 2 antibody reactivities, l to
alanyl-tRNA synthetase and the other to tRNA"'"
itself; no other antisynthetase sera are recognized to
have antibodies that bind directly to the tRNA. Thus,
the findings of Bunn and Mathews (53) may not be
pertinent to the other translation-related antibodies.
The inciting antigen relevant to myositis may be
intrinsic, i.e., the aminoacyl-tRNA synthetase itself,
perhaps after an interaction with a virus that renders it
immunogenic (12); or, particularly for the antibody to
tRNAaIa, an antiidiotypic mechanism may pertain (54).
Alternatively, a n as-yet-unidentified extrinsic protein,
perhaps viral, may mimic specific aminoacyl-tRNA
synthetases and cause an immune response via molecular mimicry (55).
Whatever the inciting immunogen(s), the data
presented here suggest that a DRpl or, less likely, a
DRp3 epitope on HLA-DR3, DR5, DRw6, and DRw8
haplotypes is necessary for T helperhducer cells to
present this antigen(s) to the appropriate B lymphocytes, to produce specific translation-related and other
myositis-related autoantibodies. Further studies
should focus on defining the specific functional
epitopes involved in the immune responses to translation-related factors, and in susceptibility t o inflammatory myositis in general.
Addendum. Since the submission of this article, an
additional 4 patients (2 white and 2 black) with polymyositis
and anti-Jo-1 have been studied similarly (by RFLP) for
HLA-DR and DQ specificities. These new patient profiles
and DR types are as follows: DR3, DRw6( 13), Dw24, Dw26
(white male with polymyositis); DR3, DR4, Dw24, DRwS3
(white female with polymyositis and interstitial lung disease): DRS, DRw6(13), Dw25, Dw24 or Dw26 (black male
with polymyositis and interstitial lung disease); and DRS,
DR2, Dw24 (black male with polymyositis and interstitial
lung disease).
ACKNOWLEDGMENTS
The authors gratefully acknowledge the technical
assistance of Denola Brown, Madeleine Gould, Karen Whittington, Miriam MacLeod, Wanda O'Brien, Edward Trieu,
and Dr. Roger Schulz, the secretarial assistance of Diane
Claude, and biostatistical advice of David Moher. We thank
Drs. D. Larhammer, E. Long, and J. Trowsdale for generously providing the probes used in this study.
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