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Polymorphism of the HLA-DMA and DMB genes in Rheumatoid Arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 40,No. 5, May 1997, pp 854-858
0 1997, American College of Rheumatology
854
POLYMORPHISM OF THE HLA-DMA AND DMB GENES IN
RHEUMATOID ARTHRITIS
VALERIEPINET, BERNARD COMBE, ODILE AVINENS, SOPHIE CAILLAT-ZUCMAN, JACQUES SANY,
JACQUES CLOT, and JEAN-FRANCOIS ELIAOU
Objective. To determine whether the HLA-DMA
and DMB genes, whose encoded molecules are involved
in HLA class II-restricted antigen presentation, contribute to the genetic susceptibility to rheumatoid arthritis (RA).
Methods. One hundred ninety-one RA patients,
147 control subjects, and 218 HLA-DRB1 genotypematched control subjects were oligotyped for DMA and
DMB genes.
Results. DMA*0103 and DMB*0104 were significantly increased in the RA patients compared with the
randomly selected and the matched controls, thus indicating a direct influence of the DM genes. After stratification of the patients and matched controls according
to DRBl genotypes, only DMA*0103 was increased in
the RA patients with DRBl*Ol, as well as in the patients
negative for the RA-susceptibility DR alleles.
Conclusion. Our results suggest that DMA*0103
could play an additional role in the genetic susceptibility to RA.
Rheumatoid arthritis (RA) is an autoimmune
disease characterized by chronic inflammatory joint
involvement. RA is also a complex, multifactorial disease with a significant genetic influence. Genes within
the HLA complex have been shown to determine susceptibility to RA, HLA-DR4 being the first to be
associated with RA (1). Advances in molecular biology
Supported in part by grants from the Association de Recherche sur la Polyarthrite and GREG (no. 88/94).
Valtrie Pinet, PhD, Odile Avinens, Jacques Clot, MD, PhD,
Jean-FranGois Eliaou, MD, PhD: Laboratoire d’Immunologie,
INSERM U291, Montpellier, France; Bernard Combe, MD, PhD,
Jacques Sany, MD: Service d’Immuno-Rhumatologie, Montpellier,
France; Sophie Caillat-Zucman, MD: HBpital Necker, INSERM U25,
Paris, France.
Address reprint requests to Jean-FranGois Eliaou, MD, PhD,
Laboratoire d’Immunologie, HBpital Saint-Eloi, Centre Hospitalier
Universitaire Montpellier, Montpellier, France.
Submitted for publication July 28, 1996; accepted in revised
form November 13, 1996.
permitted a more accurate definition of the various
HLA-DRBl alleles that confer risk: DRB1*0401,
*0404, *0405, *0408, *0101, and *0102. Although the
role of the HLA component in the susceptibility to RA
is firmly established, the underlying molecular mechanisms are still unknown. In particular, the presence of
specific amino acid motifs at positions 70-74 of the
HLA-DRP1 molecule, which determine the risk of
developing RA, cannot always account for the observed
HLA-DRB1 gene distribution in patients. This is especially true when considering the disease phenotype and
the genotype configurations of the patients (2,3).
The HLA-DMA and DMB genes are located
within the HLA class I1 region, between the DP and DQ
gene regions. The a and P glycoproteins encoded by the
DMA and DMB genes, respectively, are HLA class
II-like molecules and form a heterodimer. The
HLA-DM molecules have been localized in the endocytic compartment of the B lymphocytes, where the
peptides bind to HLA class I1 molecules (4). Cellular
mutants analyses have clearly demonstrated the direct
involvement of the DM molecules in the HLA class
II-restricted antigen presentation pathway (for review,
see ref. 5).
A limited nucleotide polymorphism has been
described in the third exon of the DMA and DMB genes
(6). This polymorphism does not involve the putative
peptide-binding pocket, but could influence either the
transport of the DM molecules across the various cellular compartments or the interaction with the DR molecules that leads to the binding of the antigenic peptides
to DR heterodimers.
To better understand the molecular basis of the
association between the HLA class I1 component and
RA, we compared the distributions of the DMA and
DMB genes in RA patients and control subjects. We
found that particular alleles at the DM loci are significantly associated with susceptibility to RA.
HLA-DMA AND DMB POLYMORPHISM IN RA
855
Table 1. Sequence of the oligonucleotides used as primers and probes for HLA-DMA and DMB gene typing
Allele detected
SOP*
Primer
*0101
HLA-DMA third exon
5'-GGGTTTCCTATCGCTGAAGTG-3'
5'-CCAATAGGCAA'TTGCTGTGTA-3'
+
+
+
+
5'J2ATCAlTCCGTCCCTGTGG-3'
5 '-CATCATTCCATCCCTGTGG-3'
5'-TGTCGATGGACTCAGCTTC-3'
5 '-TGTCGATGCACTCAGCITC-3'
5'-GAAAlTGACCGCTACACAG-3'
*0102
*0103
+
+
+
+
+
+
5 '-GAAATTGACTGCTACACAG-3'
5 '-GAAATTGACCACTACACAG-3'
HLA-DMB third exon
5 '-CGGCCACCATCTGTGCAAGT-3'
5' -CCAGTCCCGAAGGATGGGCTC-3'
+
+
5 '-GTGATGCTGGCCTGCTATG-3'
5'-AGCAGCGCGCACAAGACGT-3'
5'-AGCAGCGAGCACAAGACGT-3'
5 ' -AGCAGCGTGCACAAGACGT-3'
5 '-GTAGAGCACAITGGGGCTC-3'
5'-GTAGAGCACACTGGGGC3'
+
+
+
~
+
+
+
+
+
-I-
*0104
+
+
+
~~
*SSOP = sequence-specificoligonucleotide probe.
PATIENTS AND METHODS
Patients and controls. All patients and controls were of
Caucasian origin and were from the same geographic area. The
unrelated patient population consisted of 191 RA patients with
classic disease, according to the American College of Rheumatology (formerly, the American Rheumatism Association)
criteria (7).
Two unrelated control populations were used in this
study. All were healthy volunteer bone marrow donors. One
group consisted of 147 individuals who were randomly selected
and typed for DR and DM. To determine whether the
association between RA and certain DM alleles resulted from
a direct influence of the DM genes or an indirect influence
through linkage disequilibrium with alleles at the DRBl locus,
a second group of control subjects was defined. This group of
218 individuals was closely matched for the HLA-DRB1
genotype with the patient group.
Methods. Genomic DNA was extracted from peripheral blood mononuclear cells according to the classic saltingout procedure. DRBl alleles were determined as previously
described (8). DRBl*O4 high-resolution typings were performed by the sequencing-based typing procedure (9).
The polymorphism of the DMA and DMB genes was
determined following polymerase chain reaction (PCR) amplification of genomic DNA using 2 pairs of primers (Table 1).
Hybridization of the PCR products with the respective DMA
and DMB panel of sequence-specific oligonucleotide probes
(SSOPs) was performed using a nonradioactive direct dot-blot
procedure. The sequences of the SSOPs are given in Table 1.
Four alleles have been defined for DMA and DMB. These
alleles have been named according to the nomenclature for
factors of the HLA system (10).
Statistical analysis. Odds ratios (OR) were calculated
by the method of Woolf, with Haldane's modification for small
Table 2. Distribution of the HLA-DMA and DMB alleles in rheumatoid arthritis (RA)patients compared with the two control populations*
~~
DM gene
DMA
'0101
'0102
"0103
*0104
DMB
*0101
"0102
'0103
*0104
~
~
~
~
~
~
~
~~
No. (%)
of patients
(n = 191)
No. (%) of
random controls
(n = 147)
No. (%) of
matched controls
(n = 218)
187 (98)
29 (15)
24 (13)
2 (1)
146 (99)
33 (22)
4 (3)
4 (3)
217 (99)
43 (20)
8 (4)
l(0.5)
174 (91)
22 (12)
58 (30)
29 (15)
138 (94)
10 (7)
43 (29)
10 (7)
205 (94)
18 (8)
66 (30)
13 (6)
RA vs. RC
OR
Porr
-
NS
NS
0.008
NS
5.1
---
NS
NS
NS
NS
RC vs. MC
RC vs. MC
OR
Pcorr
OR
pcorr
-
NS
NS
0.01
NS
-
-
NS
NS
NS
NS
-
NS
3.8
--
2.8
NS
NS
NS
0.02
-
NS
NS
NS
* Control subjects were either randomty selected (RC) or were HLA-DRBl-matched (MC). The odds ratio (OR) is indicated only when the
difference is significant. NS = not significant.
PINET ET AL
RESULTS
Table 3. Distribution of DRBl*04 and DRBl*Ol genotypes in rheumatoid arthritis patients and HLA-DRB1-matched controls*
Patients
Matched controls*
89
81
7
8
3
39
5
0
19
2
6
5
3
1
47
4
2
18
0
1
37
44
4
33
6
38
DRBl*04 haplotypes
No. of study subjects
No. with DRBl genotype
*0401/*0401
*0401/*0404
*0401/*0405
*0401/*X
*0404/*0404
*0404/*0405
*04041*X
*0405/*0405
*0405/*X
DRBl*Ol haplotypes
No. of study subjects
No. with DRBl genotype
*01/*01
*Ol/*X
Association of DMA*0103 and DMB*0104 alleles
with RA. When the distribution of the DMA and DMB
alleles was compared between the RA patients and the
randomly selected controls, no difference in the distribution of DMA*0101 and DMB*0101 was seen. However, although the frequency of the DMA*0103 allele is
quite low in both populations, DMA*0103 was found to
be more frequent in the RA patients (13% in patients
versus 3% in controls, P,,, = 0.008) (Table 2). The
analysis of the distributions of the DMA and DMB
genotypes gave similar results. The genotypes including
the DMA*0103 haplotype were found to be more frequent in the patient population (data not shown).
To entirely eliminate a possible influence of a
linkage disequilibrium between alleles at the DM locus
and RA-associated DRBl alleles, the RA patients were
then compared with a group of HLA-DRB1-matched
controls. No significant difference in the genotype distribution was observed between the patients and the
matched controls (Table 3). Again DMA*0103 was
found more frequently in the patients than in the
HLA-DRB1 genotype-matched controls (13% in patients versus 4% in controls, p,,,, = 0.01) (Table 2). In
addition, the DMB*0104 allele was significantly increased in the RA patients (15% in patients versus 6% in
matched controls, P,,,, = 0.02) (Table 2). One explanation for this latter finding is that the Bonferroni inequal-
* DRBl*Ol includes DRB1*0101and *0102. None of the comparisons
was statistically significant.
numbers. The significance of differences in phenotype or
genotype frequencies was determined by Fisher's exact test.
Corrected P values (Po,,)were calculated by the Bonferroni
inequality method by multiplying P by the number of alleles
compared and, when relevant, by the number of groups. The
level of significance was set at 0.05. Associations between
HLA-DRB1 and DM alleles was assessed using the A value for
nonrandom assortment of alleles (11).
Table 4. Distribution of the HLA-DMA and DMB alleles in RA patients and HLA-DRB1-matched controls, according to the HLA-DR
genotypes*
HLA-DRBl*Ol and DRB1*04 patients and matched controls
~
DRB1*01/*01and DRBl'OlK
DMgene
DMA
'0101
*0102
*0103
*0104
DMB
'0101
'0102
*0103
*0104
No. (%) of
RA patients
(n = 37)
No. (%) of
matched
controls
(n = 44)
36 (97)
6 (16)
10 (27)
1 (3)
44 (100)
13 (30)
l(2)
0
36 (97)
8 (22)
5 (14)
6 (16)
42(95)
2 (4)
14(32)
3 (7)
DRB1'041'04
OR
P,,,,
No. (%) of
RA patients
(n = 89)
-
-
NS
NS
0.008
NS
88(99)
14(16)
6 (7)
0
-
NS
NS
NS
NS
77(87)
8 (9)
38(43)
17(19)
15.9
and DRB1'04K
No. (%) of
matched
controls
(n = 81)
HLA-DRX patients and matched controls
No. (%) of
matched
controls
(n = 78)
OR
P,,,,,
10.7
-
NS
NS
0.05
NS
-
NS
NS
NS
NS
OR
Par,
No. (%) of
RA patients
(n = 49)
SO(99)
lO(12)
5 (6)
1(1)
-
NS
NS
NS
NS
48(98)
6 (12)
6 (12)
1(2)
78(100)
16(20)
l(1)
0
74(91)
8 (10)
29 (36)
5 (6)
-
NS
NS
NS
NS
47(96)
3 (6)
11 (22)
5 (10)
74 (95)
7 (9)
18 (23)
4 (5)
-
-
-
* DRBl*Ol includes DRB1*0101and '0102. DRB1*04 includes DRB1*0401, '0404, *0405,and '0408. DRX indicates any DRBl allele except the
rheumatoid arthritis (RA)-associated DRBl*Ol and DRB1'04 alleles. The odds ratio (OR) is indicated only when the difference is significant. NS =
not significant.
HLA-DMA AND DMB POLYMORPHISM IN RA
ity method is extremely stringent and because the number of matched controls was higher than the number of
random controls, the difference compared with the RA
patients reached statistical significance. Analysis of the
genotype distributions gave similar results (data not
shown).
Another argument for the absence of linkage
disequilibrium between alleles at DM and DRBl loci is
that, although there was a significant difference in the
DRBl genotype distribution between the random controls and the matched controls (P = 0.01,J = 11.9 with
8 degrees of freedom), there was no significant difference in the distribution of DM alleles between these two
control groups (Table 2).
Combined analysis of DM and DRBl allele distributions in RA patients. It has been reported that the
genetic risk associated with the various RA-susceptibility
DRBl alleles is not equivalent (2,3). To determine
whether the difference in the distribution of the DMA
and DMB alleles could be observed among the patients
carrying the various DRBl alleles, the patients and
matched controls were stratified according to their
DRBl genotypes. Three groups were defined: patients
carrying at least one DRBl*Ol haplotype (DRB1*0101
and *0102), patients carrying at least one RA-associated
DRB1*04 haplotype (DRB1*0401, *0404, *0405, or
*0408), and patients carrying no RA-associated haplotypes (so-called DRX/DRX).
The DMA*O103 allele was found to be significantly more frequent in DRBl*Ol patients than in
controls (Table 4). This difference was seen both when
the DRBl*Ol/DRBl*Ol and the DRBl*Ol/DRX (DRX
standing for non-RA-associated haplotypes) genotypes
were considered (OR = 15.9, P,,,, = 0.008), suggesting
a combined effect of DMA*0103 and DRBl*Ol. Indeed,
in the DRBl*Ol/DRBl*Ol or DRBl*Ol/DRX genotypes, the RA-associated DRBl*O4 haplotypes are not
taken into account. Strikingly, the DMB*0104 distribution was not different in this subgroup of RA patients
compared with the matched controls.
The frequency of the DMB*0104 allele was
higher in the DRB1*04/DRB1*04 and the DRBl*O4/
DRX patients than in the matched controls. However,
the difference was not found to be significant after
correction of the P value (Table 4).
Finally, in DRX/DRX patients (Table 4), the
frequency of the DMA*0103 allele was increased compared with that in matched controls (OR = 10.7, P,,,, =
0.05), which strongly suggests that no linkage disequilibrium with the HLA-DRBl*Ol alleles could explain the
effect of DMA*0103. This was confirmed by evaluating
857
linkage disequilibrium, using A values for nonrandom
assortment of alleles in control subjects. No significant
linkage disequilibrium was found between the DRBl*Ol
and DMA*0103 alleles (A = 0.002, P = 1).
DISCUSSION
In the present study, we have shown that specific
DMA and DMB alleles are associated with RA susceptibility. The influence of the DMA and DMB alleles
cannot be explained by a linkage disequilibrium with
RA-associated HLA-DRBl alleles. This was demonstrated both by the use of an HLA-DRB1 genotypematched control population and by determination of the
A value for nonrandom assortment of alleles in controls.
The risk associated with the DMB*0104 allele
can only be observed when the global population of
patients is considered, which suggests that this allele has
only a minor effect on genetic susceptibility to RA.
However, DMA*0103 seems to play an important role in
disease susceptibility, since it was more frequent both in
the overall RA population as well as in the DRBl*Ol
haplotype-positive patients. It is difficult, at the present
time, to precisely define the impact of DMA*0103 on
the genetic susceptibility to RA. However, our data
suggest that DRBl*Ol and DMA*0103 alleles interact,
although DMA*0103 contributes independently of
DRBl*Ol to the genetic risk of RA in DRX/DRX
patients.
Patients with DRB1*04, and especially those with
DRB1*04/DRBl*04, are known to have a higher risk
of developing severe disease than are patients with
DRBl*Ol or DRX (3). The fact that DMA*0103was not
found to be increased in patients with the DRB1*04
haplotype might suggest that this allele could be associated with the progression and severity of RA in patients
with DRBl*Ol and DRX. The combined presence of
DMA*0103 and DRBl*Ol or DRX could represent a
bad prognosis factor in patients expected to be at low
risk of developing a severe form of RA. This has yet to
be confirmed by prospective clinical and immunogenetic
studies.
Although the functional consequences of the
polymorphism of the DM genes have not yet been
demonstrated, it could be suggested that the class 11restricted antigen-presentation pathway involving the
DM molecules could play a role in the pathophysiology
of the disease. This may represent an additional molecular mechanism of the association between the HLA
complex and RA.
PINET ET AL
858
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