Human leukocyte antigen-DRB1 position 11 residues are a common protective marker for sarcoidosis.

Abstract

Genetic factors, in particular human leukocyte antigens (HLAs) are important determinants of susceptibility to sarcoidosis, a chronic granulomatous disease of undetermined etiology. To clarify the role of HLA in sarcoidosis we determined HLA-DR and -DQ alleles in case-control samples from three European populations (United Kingdom, Czech, and Polish) and compared these results with those published for three additional populations (Italian, Japanese, and Scandinavian) to determine whether the HLA-DR and/or -DQ alleles act as ethnic-dependent, or ethnic-independent modifiers of disease risk. Although variations were apparent in the alleles associated with susceptibility, reductions in the frequency of alleles associated with protection were remarkably consistent in the six populations. Previously detected associations between single-nucleotide polymorphisms at the TAP2 locus and sarcoidosis were shown to be due to linkage disequilibrium with the HLA-DR locus. The protective HLA-DR alleles, which encode the DR1 and DR4 antigens, were found to share characteristic small hydrophobic residues at position 11, which were replaced by small hydrophilic residues in the remaining, nonprotective, HLA-DR alleles. This residue position is within a pocket of the HLA-DR complex antigen binding groove (designated P6), where it is the only variable amino acid and therefore determines the peptide binding preferences of this pocket. A highly significant reduction in the frequency of individuals carrying HLA-DR alleles with a hydrophobic residue at position 11 was observed in the sarcoidosis cases in the three populations we examined. This suggests this HLA-DR residue is an important protective marker in sarcoidosis.

Full text

Page 1

Am. J. Respir. Cell Mol. Biol.
Internet address: www.atsjournals.org
Vol. 25, pp. 272–277, 2001
Human Leukocyte Antigen-DRB1 Position 11 Residues Are a Common
Protective Marker for Sarcoidosis
Patrick J. Foley, Deirdre S. McGrath, Elzbieta Puscinska, Martin Petrek, Vitezslav Kolek, Jiri Drabek,
Penelope A. Lympany, Panagiotis Pantelidis, Kenneth I. Welsh, Jan Zielinski, and Roland M. du Bois
Interstitial Lung Disease Unit, Department of Occupational and Environmental Medicine, National Heart and Lung Institute, Imperial
College of Science, Technology, and Medicine, London; Oxford Tissue Typing Centre, Nuffield Department of Surgery, The Churchill
Hospital, Oxford, United Kingdom; Institute of Tubercle and Lung Diseases, Warsaw, Poland; Department of Immunology, Medical
Faculty of Palacky University; and Department of Respiratory Medicine, University Hospital, Olomouc, Czech Republic
Genetic factors, in particular human leukocyte antigens (HLAs)
are important determinants of susceptibility to sarcoidosis, a
chronic granulomatous disease of undetermined etiology. To
clarify the role of HLA in sarcoidosis we determined HLA-DR
and -DQ alleles in case-control samples from three European
populations (United Kingdom, Czech, and Polish) and com-
pared these results with those published for three additional
populations (Italian, Japanese, and Scandinavian) to deter-
mine whether the HLA-DR and/or -DQ alleles act as ethnic-de-
pendent, or ethnic-independent modifiers of disease risk. Al-
though variations were apparent in the alleles associated with
susceptibility, reductions in the frequency of alleles associated
with protection were remarkably consistent in the six popula-
tions. Previously detected associations between single-nucle-
otide polymorphisms at the TAP2 locus and sarcoidosis were
shown to be due to linkage disequilibrium with the HLA-DR
locus. The protective HLA-DR alleles, which encode the DR1
and DR4 antigens, were found to share characteristic small hy-
drophobic residues at position 11, which were replaced by
small hydrophilic residues in the remaining, nonprotective,
HLA-DR alleles. This residue position is within a pocket of the
HLA-DR complex antigen binding groove (designated P6),
where it is the only variable amino acid and therefore deter-
mines the peptide binding preferences of this pocket. A highly
significant reduction in the frequency of individuals carrying
HLA-DR alleles with a hydrophobic residue at position 11 was
observed in the sarcoidosis cases in the three populations we
examined. This suggests this HLA-DR residue is an important
protective marker in sarcoidosis.
Sarcoidosis, a disease predominantly affecting young adults,
is the most common diffuse lung disease, with a population
prevalence of around 1:4,000 in the United Kingdom
(UK). Increased disease risk among patients’ families and
varying racial disease incidences suggest that genetic fac-
tors are important determinants of susceptibility to this
chronic granulomatous disease of undetermined etiology
(1). In sarcoidosis, CD4
?
T cells accumulate at the site of
active disease, where they are believed to interact via their
T-cell receptor for antigen with human leukocyte antigen
(HLA) molecules expressed by antigen-presenting cells
(APCs). Expression of HLA-DR on alveolar macrophages
from pulmonary sarcoidosis patients has been shown to be
increased (2), as has the antigen-presenting capacity of
these cells (3). These findings suggest that in sarcoidosis,
antigen presentation by APCs occurs via the HLA class II
restricted pathway, and that this process is likely to be cen-
tral to the initiation and maintenance of the granulomatous
inflammation observed.
Several studies have examined major histocompatibility
complex (MHC) class II alleles in sarcoidosis, but have not
identified strong associations consistently (4, 5) possibly
reflecting small sample sizes, involvement of nearby “non-
classical” genes within the MHC region, and ethnic, or
clinical, heterogeneity in subjects studied. Use of multiple
clearly defined ethnic populations may therefore be a use-
ful strategy when performing studies of this disease. We
previously examined whether specific alleles or allelic mo-
tifs (residues common to multiple alleles) at the HLA-
DPB1 locus contribute to sarcoidosis susceptibility in a
case-controlled study of the UK population (6). No evi-
dence of associations with specific alleles was detected at
this locus, in agreement with findings in African-American
patients (7). No allelic motif associations were observed in
the UK patients, although the Val36- and Asp55-positive
HLA-DPB1 alleles were associated with increased disease
risk in the previous African-American patient study. In
our previous study, associations were detected between
sarcoidosis and single-nucleotide polymorphisms (SNPs)
in the TAP2 antigen-processing gene, located within the
MHC class II region, in case-control samples from both
the UK and Polish populations. SNPs at the nearby TAP1
gene showed no association with disease. The lack of asso-
ciation between polymorphic loci located centromeric to
the TAP2 gene (i.e., the HLA-DP and TAP1 loci) suggests
that the TAP2 gene may represent a boundary of the
MHC region involved in sarcoidosis susceptibility. In the
present study, we examined allelic variation at the HLA-
DR and -DQ loci, which are telomeric to the TAP2 loci, to
clarify further the exact role this MHC region plays in de-
termining susceptibility to sarcoidosis. An association be-
tween a particular HLA allele and disease across different
ethnic groups lends support to the direct participation of
that locus in disease susceptibility (8). To identify whether
any relationships between alleles and disease in sarcoido-
sis are consistent across ethnic boundaries, we studied
three groups of case-control samples from the UK Cauca-
sian, Polish, and Czech populations.
(
Address correspondence to:
ease Unit, Dept. of Occupational & Environmental Medicine, National
Heart & Lung Institute, Imperial College of Science, Technology & Medi-
cine, London, SW3 6LR, UK. E-mail: r.dubois@rbh.nthames.nhs.uk
Abbreviations:
?
-square,
?
; 95% confidence interval, 95% CI; degrees of
freedom, d.f.; human leukocyte antigen, HLA; hydrophobic side chain,
HS; major histocompatibility complex, MHC; nonhydrophobic side chain,
NHS; odds ratio, OR; polymerase chain reaction, PCR; single-nucleotide
polymorphism, SNP.
Received in original form June 6, 2000 and in revised form March 27, 2001
Professor R.M. du Bois, Interstitial Lung Dis-
)
2

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Foley, McGrath, Puscinska,
et al
.: HLA-DRB1 Position 11 Residues Are Protective in Sarcoidosis273
Materials and Methods
Subjects
Patients at the three participating study centers were defined us-
ing the ATS/ERS/WASOG Statement on Sarcoidosis (9). All pa-
tients were diagnosed on the basis of noncaseating granulomas in
a tissue biopsy and had clinical features of pulmonary sarcoidosis.
UK cases (
n

?
189) were recruited from the Royal Brompton
Hospital, London, UK; Czech cases (
University Hospital, Olomouc, Czech Republic; and Polish cases
(
n

?
87) from the Institute of Tubercle and Lung Diseases, War-
saw, Poland. Unrelated, ethnically matched controls were Cauca-
sian cadaveric organ donors from the Oxford Tissue Typing Cen-
tre (
n

?
288), Oxford, UK; Czech nationality blood donors from
the Olomouc region (
n

?
158); and healthy medical students
from the Medical University of Gdansk, Gdansk, Poland (
133). Patient demographics, including age at presentation, sex,
and smoking history are outlined in Table 1. Part of the UK case
population (
n

?
117) and all of the UK controls and Polish cases
and controls were included in our previous study of different
genes (6). Genotype data on the TAP genes from our previous
study is referred to for linkage. Project approval was granted by
the local ethics committee at each of the three centers.
n

?
69) from the Olomouc
n

?
HLA-DR and -DQ Genotyping
Genomic DNA was isolated from whole-blood samples accord-
ing to Miller and colleagues (10). A 48-reaction sequence-specific
primer (SSP) polymerase chain reaction (PCR) assay was used to
determine the HLA-DR and -DQ genotypes for each individual
studied. PCR reaction mixture components, cycling parameters,
and the detection of PCR products by agarose gel electrophoresis
were as described previously (11).
Comparison of HLA Results with Published Data
To determine whether any alleles represent ethnic-independent
susceptibility or protection factors, the results for the populations in
this study were compared with those published previously for case-
control studies from Scandinavia, Italy, and Japan (12–14). Studies
used for comparison were chosen on the basis of size (all three stud-
ies examined in excess of 100 cases, and 250 matched controls) and
provision of a medium resolution HLA-DR phenotype frequency
breakdown. In the case of the Japanese population, several HLA-
DR studies have been described; the study chosen for comparison
represented the largest of these. In the previously published studies
used in the comparisons, HLA data was obtained using both sero-
logic and SSP-PCR methodology. These methodologies conformed
to the World Health Organization (WHO)–defined standards for
HLA nomenclature and typing used here.
After comparison of the results, HLA-DR alleles were classed
as “susceptible” or “protective” if the phenotype frequency was
increased or decreased, respectively, in patients compared with
matched controls.
We also compared the results of HLA-DR and -DQ genotyp-
ing with our previous TAP2 genotyping results for the UK and
Polish patient groups (6) to determine whether associations ob-
served at the HLA-DR and -DQ loci in the present study occurred
independently of those observed previously at the TAP2 locus.
HLA-DRB1 Protein Sequence Comparisons
Protein sequences of HLA-DRB1 alleles were obtained from the
IMGT/HLA Database (15). The relative positions of HLA-DRB1
TABLE 1
Demographic data on UK, Polish, and Czech sarcoidosis patients
UK SarcoidPolish Sarcoid Czech Sarcoid
FM TotalFM TotalFM Total
Number (
Age at presentation (yr)
Mean
Standard error of the mean
Smoker?
Yes
No
n
) 89100189 4839 87 3831 69
40
10
39
8
39
9
53
9
44
7
49
9
48
9
42
8
45
11
35837 10
77
257
84 97 1814532 3626 62
TABLE 2
Phenotype frequencies (%) of HLA-DR and -DQ alleles
in sarcoidosis cases and controls from
three European populations
UKPolish Czech
Allele
Cases
n
= 189)(
Controls
(
n
= 288)
Cases
n
= 87)(
Controls
(
n
= 133)
Cases
n
= 69)(
Controls
(
n
= 158)
DRB1*01
DRB1*04
DRB1*07
DRB1*08
DRB1*09
DRB1*10
DRB1*11
DRB1*12
DRB1*13
DRB1*14
DRB1*15
DRB1*16
DRB1*17
DQB1*02
DQB1*04
DQB1*05
DQB1*0601
DQB1*0602
DQB1*0603-9
DQB1*0301/4
DQB1*0302
DQB1*03032
11.2
25.5
19.7
3.2
1.6
2.7
17.6
10.1
17.6
16.5
38.8
0.5
25
37.8
2.7
29.3
0
38.6
3.7
36.2
12.8
6.4
19.3*
35.0*
26.7
4.8
2.5
2.3
12.5
2.7*
19.3
4.7*
27.9*
1.2
26.1
43.1
5.9
32.3
0.4
23.3*
16.6*
26.4*
18.4
3.1
10.3
23
23
12.6
2.3
0
31
2.3
19.5
5.7
33.3
4.6
21.8
36.8
12.6
20.7
0
32.2
11.5
36.8
18.4
9.2
29.3*
21.1
25.6
6.8
0.8
2.3
23.3
5.3
18.8
2.3
18.8*
7.5
24.1
40.6
5.3
41.4*
0
18.0*
3.0*
38.3
14.3
3
10.1
15.9
10.1
7.2
1.4
0
20.3
2.9
26.1
15.9
34.8
7.2
33.3
47.8
8.7
33.3
0
39.1
11.6
26.1
13
1.4
16.5
17.1
25.9*
3.2
0.6
0
22.2
4.4
18.3
6.3*
29.7
12
27.2
27.8*
2.5*
25.9
0
30.4
5.7
25.9
8.9
3.1
*Significant differences in phenotype frequency between cases and controls.
Individual
P
values and confidence intervals for these differences are detailed
in R
ESULTS
.

Page 3

274
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 25 2001
residues forming pockets within the peptide binding groove of the
HLA-DR complex were identified from crystallography data for
a HLA-DR peptide complex (16).
Statistical Analysis
Statistical analysis of the HLA frequency data for the three pop-
ulations studied used a
?
-square (
with the appropriate number of degrees of freedom. Significant
differences between cases and controls were determined by a
value of less than 0.05. Combined analysis of data from the three
centers used the Mantel–Haenszel test for association, with strat-
ification according to ethnic group, after testing to exclude heter-
ogeneity between the centers. Analysis of linkage disequilibrium
and standardized
?
values (
?
S) for associations between TAP2
and HLA-DR alleles were as previously described (17).
In case-control studies where the disease is rare in the general
population from which cases and controls are selected, such as
sarcoidosis, the odds ratio (OR) may be used as an approximation
of the relative risk. ORs and 95% confidence intervals (95%CIs)
were calculated for the phenotypes and genotypes found to differ
significantly. Our case-control sample sizes allowed us to detect a
difference of 10% in phenotype or genotype frequency with
93.1% power (
P

?
0.05) in the UK population, with 63% power in
the Polish population (
P

?
0.05), and with 61% power in the
Czech population (
P

?
0.05). If an ethnic-independent genetic ef-
fect was present, the combined patients and controls would, theo-
retically, detect a 10% increase in exposure within the patient
group with 98.5% power (
P

?
0.01). Statistical tests were per-
formed using the statistical program EpiInfo6 (WHO, Geneva,
Switzerland/Centers for Disease Control, Atlanta, GA).
?
2
) contingency table analysis
P
Results
HLA-DRB1 and -DQB1 Phenotypes in Three European
Case-Control Populations
The HLA-DRB1 and -DQB1 phenotype results for the UK,
Polish, and Czech cases and controls are given in Table 2.
In the UK population, an increased risk of disease was as-
sociated with the HLA-DRB1*12 (
95%CI 1.59 to 10.03), -DRB1*14 (
95%CI 1.91 to 7.9), and -DRB1*15 (
95%CI 1.10 to 2.49) alleles at the HLA-DR locus; and
with the HLA-DQB1*0602 (
95%CI 1.37 to 3.18) and -DQB1*0301/4 (
1.58, 95%CI 1.04 to 2.4) alleles at the HLA-DQ locus. A
reduced risk of disease was found to be associated with the
HLA-DRB1*01 (
P

?
0.02, OR
0.92), -DRB1*04 (
P

?
0.04, OR
0.97), and HLA-DQB1*0603-9 (
95%CI 0.08 to 0.45) alleles in the UK population.
In the Polish population, HLA-DRB1*15 (
2.16 95%CI 1.11 to 4.22) and -DQB1*0602 (
2.20, 95%CI 1.12 to 4.25) were associated with increased
risk of disease, and HLA-DRB1*01 (
95%CI 0.12 to 0.64), -DQB1*05 (
95%CI 0.19 to 0.72), and -DQB1*0603-9 (
4.19, 95%CI 1.15 to 16.49) were associated with a reduced
risk of disease. In the Czech population, an increased dis-
ease risk was associated with the HLA-DRB1*14 (
OR
?
2.8, 95%CI 1.04 to 7.6), -DQB1*02 (
2.4, 95%CI 1.3 to 4.5), and -DQB1*04 (
3.67, 95%CI 0.9 to 16.1) alleles. The HLA-DR7 allele was
associated with a reduced risk of disease (
0.32, 95%CI 0.12 to 0.8) in the Czech population.
The close proximity of the TAP2 gene, where we previ-
ously described associations in sarcoidosis (6), to the HLA-
DR and -DQ genes suggested that associations at the two
loci may not be independent, due to linkage disequilib-
rium between the loci. Data from a study of 115 homozy-
gous typing cell lines has suggested that HLA-DRB1*01
may be in linkage disequilibrium with the TAP2*0201 al-
lele (which comprises three TAP2 variant residues Val379-
P

?

?
0.00004, OR
P

?
0.02, OR
0.001, OR
?
?
?
3.93,
3.86,
1.65,
P
P

?
0.0003, OR
P
?
2.09,

?
0.03, OR
?
?
?

?
0.52, 95%CI 0.29 to
0.63, 95%CI 0.41 to
0.00001, OR
P
?
0.19,
P
P

?
?
0.02, OR
0.02, OR
?
?

P

?

?
0.002, OR
P

?
0.002, OR
?
?
0.28,
0.37,
P
0.01, OR
?
P

?
0.02,
P
P

?
?
0.003, OR
0.04, OR
?
?

P

?
0.007, OR
?
TABLE 3
Linkage disequilibrium between HLA-DRB1 and TAP2
in UK and Polish cases studied
DRB1*01
?
DRB1*01
??
S
?
2
P
*
UK cases
(
n

?
132)
Polish cases TAP2*0201
(
n

?
87)
TAP2*0201
TAP2*0201
?
?
1129
88
27
51
0.61 14.8 0.0001
4
7
2
?
?
0.636.3 0.01
TAP2*0201
*Fisher’s Exact test used for the Polish comparison.
TABLE 4
Consistently protective HLA-DRB1 alleles in sarcoidosis
Phenotype FrequencyOR (95%CI)*
Population
n
DRB1*01DRB1*04DRB1*01DRB1*04
UK cases
UK controls
Czech cases
Czech controls
Polish cases
Polish controls
Scandinavian cases (12)
Scandinavian controls
Japanese cases (14)
Japanese controls
Italian cases (13)
Italian controls
189
288
69
158
87
133
122
250
113
478
107
510
0.112
0.193
0.101
0.165
0.103
0.293
0.130
0.180
0.009
0.098
0.065
0.165
0.255
0.350
0.159
0.171
0.230
0.211
0.210
0.370
0.336
0.435
0.074
0.135
0.52 (0.29–0.92)0.63 (0.41–0.97)
0.41 (0.14–1.11)0.92 (0.40–2.10)
0.28 (0.12–0.64)1.02 (0.51–2.05)
0.69 (0.35–1.32)0.46 (0.27–0.78)
0.08 (0–0.56)0.66 (0.42–1.03)
0.40 (0.16–0.92)0.52 (0.22–1.16)
*ORs for the HLA-DRB1*01 and -DRB1*04 alleles in each population were calculated on the risk conferred by the presence of one or more copies of that allele.

Page 4

Foley, McGrath, Puscinska,
et al
.: HLA-DRB1 Position 11 Residues Are Protective in Sarcoidosis 275
Ala565-Ala665) (18). This association was confirmed in
our UK and Polish patient populations (Table 3). The fre-
quency of the TAP2 variants Val379 and Ala665 were
found to be significantly reduced in the previous UK and
Polish population study, and these reductions correlate
with the reductions in the HLA-DRB1*01 frequency seen
in the present study.
Comparison of HLA Phenotype Results with
Published Data
Results for the three populations in this study were com-
pared with results previously published for Scandinavian,
Italian, and Japanese case-control studies. The “suscepti-
ble” alleles were found to vary between populations, but
reductions in the frequency of “protective” alleles, encod-
ing the DR1 and DR4 antigens, were remarkably consis-
tent in the six populations (Table 4).
Sequence Comparison of “Susceptible” and
“Protective” Alleles
To identify common features exclusive to either the “pro-
tective” (HLA-DRB1*01 and -DRB1*04) or the “suscepti-
ble” (HLA-DRB1*08, -DRB1*09, DRB1*12, -DRB1*14, -
DRB1*15, and -DRB1*17) allele groups, protein sequences
of the HLA-DRB1 alleles were compared. One such resi-
due was identified: in the “protective” alleles, at position 11,
a valine or leucine was present, whereas in the “susceptible”
alleles a glycine, serine, or proline was found at this posi-
tion. Structurally, valine and leucine are small residues and
have bulky, highly hydrophobic, aliphatic side chains,
whereas serine, glycine, and proline are small and do not
have highly hydrophobic side chains. The remaining HLA-
DRB1 alleles were also found to contain small, hydrophilic
residues at position 11. The peptide binding specificity of
HLA-DR is determined by clusters of polymorphic residues
which form nine pockets in the HLA-DR groove (desig-
nated P1 through P9) (16). The variable residues confer dis-
tinct chemical and size characteristics to each pocket in dif-
ferent HLA-DRB1 alleles (19). Within the P6 cluster,
HLA-DRB1 position 11 is the only variable residue and
therefore determines the binding preferences of this pocket.
Influence of HLA-DRB1 Position 11 Side-Chain Properties
on Susceptibility to Sarcoidosis
To determine the significance of hydrophobic or hydro-
philic side-chain residues at position 11 of the HLA-DRB1
peptide in sarcoidosis, the HLA-DR results were reclassi-
fied into “hydrophobic side-chain” (HS) and “nonhydro-
phobic side-chain” (NHS) alleles based on the residue 11
side-chain properties. These results were then compared
among our three study populations (Table 5). Analysis of
the reclassified data showed a significant negative associa-
tion between sarcoidosis and DRB1 genotypes and alleles
in both the UK (?2 ? 20.3, degrees of freedom (d.f.) ? 2,
P ? 0.00004, and ?2 ? 20.8, d.f. ? 1, P ? 0.000005, respec-
tively) and Polish (?2 ? 7.7, d.f. ? 2, P ? 0.02, and ?2 ?
5.1, d.f. ? 1, P ? 0.02, respectively) samples. The variance
in genotypic distribution appeared attributable to a reduc-
tion in the HLA-DRB1 HS alleles in the sarcoidosis cases
(UK: P ? 0.0001, OR ? 0.46, 95%CI ? 0.31 to 0.69; Pol-
ish: P ? 0.008, OR ? 0.46, 95%CI ? 0.25 to 0.85). In the
Czech sample, a similiar reduction was detected but did
not reach significance when tested (OR ? 0.79, 95%CI ?
0.40 to 1.55). Combining the three study samples in a strat-
ified analysis showed that the sarcoidosis cases had a
highly significant reduction in the HLA-DRB1 HS alleles
(?2 ? 24.6, d.f. ? 2, P ? 0.0000007). This yielded a Mantel–
Haenszel weighted OR of 0.56 (0.44 to 0.7), with no evi-
dence of heterogeneity between samples (P ? 0.33).
Discussion
In this study we examined HLA-DR and -DQ allele distribu-
tions in three European case-control samples to clarify the
role that the MHC class II region plays in determining sus-
ceptibility to sarcoidosis. Our use of the words “population,”
“ethnic,” and “ethnic boundaries” in referring to these three
groups is intended to convey that the three case-control sam-
ples chosen have genetic backgrounds that are distinct from
each other. That the UK, Polish, and Czech samples are in
fact genetically different is apparent from the HLA distribu-
tion in the control groups from each country that show clear
differences, in agreement with published HLA data for the
English, Polish, and Czech populations which also show dif-
ferences in HLA distribution between these groups (15). Re-
sults for the three populations examined (UK, Polish, and
Czech) were further compared with those published for three
additional populations (Italian, Japanese, and Scandinavian)
to determine whether the HLA-DR and/or -DQ alleles act as
ethnic-dependent, or ethnic-independent modifiers of dis-
ease risk. Although variations were apparent in the alleles as-
sociated with susceptibility, reductions in the frequency of al-
leles associated with protection were remarkably consistent
in the six populations. These protective alleles, which encode
the DR1 and DR4 antigens, were found to share characteris-
tic small hydrophobic residues at position 11, which were re-
placed by small hydrophilic residues in the remaining, non-
TABLE 5
HLA-DRB1 hydrophobic position 11 alleles in sarcoidosis
Genotypes Allele OR
HS HS HS NHS NHS NHS HS NHS(95%CI)*
UK cases8 61120
(0.63)
126
(0.44)
50
(0.72)
110
(0.70)
62
(0.71)
71
(0.53)
234
(0.68)
307
(0.54)
77301
(0.04)
34
(0.12)
1
(0.01)
10
(0.06)
7
(0.08)
12
(0.09)
16
(0.04)
56
(0.10)
(0.32)
128
(0.44)
18
(0.26)
38
(0.24)
18
(0.21)
50
(0.38)
96
(0.28)
210
(0.37)
(0.20) (0.80)
190
(0.34) (0.66)
20
(0.14) (0.86)
58
(0.18) (0.82)
32
(0.18) (0.82)
74
(0.28) (0.72)
128
(0.18) (0.82)
322
(0.28) (0.72)
0.46
UK controls374 (0.31–0.69)
Czech cases 118
0.79
Czech controls258 (0.4–1.55)
Polish cases142
0.46
Polish controls 192(0.25–0.85)
Total cases564
0.55
Total controls824 (0.43–0.70)
*Numbers of cases and controls are as in Table 1. ORs for each population
and for the total samples were calculated on the risk conferred by the presence
of at least one of the HS alleles.

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276
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 25 2001
protective HLA-DR alleles. This residue position is within a
pocket of the HLA-DR complex antigen binding groove
(designated P6) (16), where it is the only variable amino acid
and therefore determines the peptide binding preferences of
this pocket (19). A highly significant reduction in the fre-
quency of individuals carrying HLA-DR alleles with a hydro-
phobic residue at position 11 was observed in the sarcoidosis
cases in the three populations we examined. This finding sug-
gests that this HLA-DR residue is an important protective
marker in sarcoidosis.
The results of this study indicate that MHC associations
in sarcoidosis reflect involvement of HLA-DR rather than
other class II loci. This is suggested by our finding that as-
sociations detected between SNPs at the TAP2 locus are
due to linkage disequilibrium with the HLA-DR1 allele in
both the UK and Polish populations. Involvement of the
HLA-DQ locus is also unlikely, considering that the pres-
ence of a specific HLA-DR allele on a haplotype, rather
than a HLA-DQ allele, appears to determine disease sus-
ceptibility. For example, the HLA-DQB1 allele -DQ5 is in
strong linkage disequilibrium with the HLA-DRB1 alleles
-DR1 and -DR14 in Caucasians. The HLA-DR1 and -DR14
alleles were found to be associated with disease “protec-
tion” and “susceptibility,” respectively, in three European
populations studied, suggesting that the HLA-DR allele
rather than the -DQ allele is more influential in determin-
ing disease risk. Of interest also was the observation that
HLA-DR14 and -15 were more common in the cases from
the three European populations we examined, whereas
the frequency of -DR17 was not altered. In the Scandina-
vian population, all three alleles were found to be associ-
ated with disease, with HLA-DR17 particularly associated
with an acute disease course; whereas -DR14 and -DR15
were strongly associated with a chronic disease course (12).
A similiar association between HLA-DR17 and acute dis-
ease has been described in German sarcoidosis cases (20).
The absence of such a finding in our three study popula-
tions may reflect a higher proportion of patients with chronic
disease in certain populations. The availability of disease
course data on our study populations will enable us to fur-
ther clarify this possibility in the future.
Our findings that certain HLA-DR alleles act as pro-
tective markers in sarcoidosis in a majority of populations
examined suggest that this protective effect is ethnic-inde-
pendent. In contrast, HLA-DR alleles associated with sus-
ceptibility appear to act in an ethnic-dependent manner, in
agreement with previous findings (12, 14). For example,
the HLA-DRB1*08 and -DRB1*09 alleles were relatively
uncommon and not strongly associated with sarcoidosis in
the three European populations we analysed, but were the
most common alleles in Japanese patients and were
strongly associated with disease (14). Conversely, HLA-
DRB1*07 was found to be present at reduced frequency in
the patient groups of the three populations examined in
the current study (UK, Polish, and Czech), but at in-
creased frequency in Scandinavian patients (12) and ab-
sent in Japanese patients and controls (14) examined pre-
viously. All of these alleles (HLA-DRB1*07, -DRB1*08,
and -DRB1*09) carry hydrophilic residues at position 11,
however their differing influences on disease susceptibility
within specific populations suggest interactions between
other elements within the HLA-DR complex in addition
to the P6/residue 11 interaction proposed here. This is sup-
ported by the high frequency of individuals carrying HLA-
DR alleles with hydrophilic residues at position 11 in our
three control populations (88 to 94%). Absence of a hy-
drophobic residue at position 11 of HLA-DR alleles can-
not therefore be considered as a discriminating feature of
susceptibility associated alleles, inasmuch as such alleles
are carried by a majority of the healthy population. In con-
trast, the presence of a hydrophobic residue at position 11
does discriminate protective alleles, and this is supported
by the increased frequency of such alleles in the control
groups from our three study populations (present in 30 to
55% of controls compared with 26 to 36% of cases).
The basis of a protective effect of hydrophobic residues
at HLA-DRB1 position 11 in sarcoidosis is unknown, but
may involve direct interaction with the peptide side chain
or indirect means. Crystallography data indicates that the
P6 pocket can accommodate water molecules concurrently
with a side chain from a MHC-bound peptide (16). Because
H2O molecules can be important in binding, the presence
of a hydrophobic position 11 side chain may alter the num-
ber/position of H2O molecules in this pocket, thereby indi-
rectly influencing the strength of the MHC binding of the
peptide(s) involved in sarcoidosis pathogenesis. Our ob-
servation that HLA-DRB1*01 and -DRB1*04 phenotype
frequencies are reduced in sarcoidosis cases in the major-
ity of populations tested suggests, for the first time, that a
common protective mechanism exists. Whether the appar-
ent protective effect of HLA-DRB1 alleles with a hydro-
phobic position 11 residue is due to altered binding of a
foreign antigen or an autoantigen is unclear, as the disease
causing antigenic stimulus remains undetermined.
Acknowledgments: The authors thank Dr. K. Koss for assistance in collecting
the Polish control sample. One author (P.J.F.) was supported by the Stanley
Thomas Johnson Foundation, Switzerland. Participation of the Olomouc inves-
tigators was supported by grant aid from the Czech Government (CEZ J14/98
151100002).
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