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Clinical Endocrinology (2002) 56, 773777
2002 Blackwell Science Ltd 773
BlackwellScience,Ltd
Preliminary evidence that an endogenous retrovirallong-terminal repeat (LTR13) at the HLA-DQB1 gene
locus confers susceptibility to Addisons disease
Michael A. Pani*, Christian Seidl, Katrin Bieda*,
Jochen Seissler, Maren Krause*, Erhard Seifried,
Klaus-H. Usadel* and Klaus Badenhoop*
*Department of Internal Medicine I, University Hospital,
Frankfurt am Main, Red Cross Blood Donor Center,
Frankfurt am Main, German Diabetes Research Institute,
Heinrich Heine University, Dsseldorf, Germany
(Received 18 December 2001; returned for revision 11 January
2002; finally revised 6 February 2002; accepted 5 March 2002)
Summary
OBJECTIVEAddisons disease is associated withparticular haplotypes of the human leucocyte antigen
(HLA) region [DQA1*0501-DQB1*0201 (DQ2) and
DQA1*0301-DQB1*0302 (DQ8)]. This locus harbours
several human endogenous retroviral (HERV) long-
terminal repeats (LTR). LTRs within the HLA region
have been shown to confer additional susceptibility to
type 1 diabetes and rheumatoid arthritis.
DESIGNWe investigated the role of LTR3 and LTR13,both of which are located adjacent to the DQB1 gene,
in Addisons disease.
PATIENTSEighty-seven patients and 160 controlswere genotyped for HLA-DQA, -DQB, and the presence
or absence of LTR3 and LTR13.
RESULTSSignificantly more patients HLA allelesthan those of controls carried the LTR13 insertion
(190% vs. 106%, P= 00143), whereas there was
only a trend for LTR3 (allele-wise chi-squared test:
P= 00941). Both, LTR3 and LTR13 are in strong link-
age disequilibrium with DQ8, which itself was signi-
ficantly more frequent in patients than in controls
(299% vs. 150%, P= 00089). However, significantlymore alleles of DQ8
+patients than of DQ8
+controls
carried the LTR13 insertion (442% vs. 188%, P=
00119), whereas we did not observe any difference for
LTR3 in the DQ8+
subset (305 vs. 231%, P= 09416).
CONCLUSIONSWe have found preliminary evidencethat the endogenous retroviral element DQ-LTR13,
but not LTR3, is associated with Addisons disease.
LTR13 appears to enhance HLA-DQ8 mediated dis-
ease risk. This retroviral insertion therefore might
represent a novel susceptibility factor in Addisons
disease, but these findings need to be confirmed in a
larger data set.
Addisons disease is a rare autoimmune disorder affecting the
adrenal gland. Currently, it is the most common cause of primary
adrenal failure with a prevalence of 40110 cases per 1 million
inhabitants (Kong & Jeffcoate, 1994; Laureti et al., 1999).
Approximately 5060% of patients with Addisons disease
develop additional autoimmune endocrinopathies during their
life as manifestations of polyglandular syndrome type 1 or 2
(Ahonen et al., 1990; Betterle et al., 1996). Genetic susceptibil-
ity conferred by a DQ gene within HLA region (HLA-DQ) is
shared in type 1 diabetes, Graves disease, Hashimotos thyroi-
ditis and Addisons disease (Badenhoop et al., 1995). Compared
to other endocrine autoimmune disorders, little is known aboutthe mechanisms involved in the pathogenesis of Addisons
disease (Betterle & Volpato, 1998; Peterson et al., 2000). Addisons
disease is thought to be mainly T cell mediated (Nerup &
Bendixen, 1969; Fairchildet al., 1980), and it is strongly associated
with the major histocompatibility complex on chromosome 6.
Both human leucocyte antigen (HLA) class II haplotypes,
DR4-DQ8 and DR3-DQ2 (Badenhoop et al., 1995), as well as
polymorphisms within the cytotoxic T lymphocyte antigen 4
(CTLA4) gene (Donneret al., 1997; Kemp et al., 1998), have been
shown to confer disease susceptibility. Autoantibodies to adrenal
cortex and 21-hydroxylase can be found in 81% of autoimmune
Addisons patients (Betterle et al., 1999) and autoantibody levels
were shown to correlate with the degree of adrenal dysfunction
in individuals with preclinical disease (Laureti et al., 1998).
Long-terminal repeats (LTRs) are common retrovirus related
sequences spread throughout the human genome. LTRs contain
regulatory sequences reported to influence the expression of
adjacent cellular genes possibly playing a role in the pathogenesis
of autoimmunity. Two LTRs, approximately 1000 bp in length,
of a human endogenous retrovirus type K (HERV-K), are located
15 kb upstream of HLA DQB1 (Kambhu et al., 1990). Previously,
we reported a human-specific integration of one of these LTRs
Correspondence: Dr Klaus Badenhoop, Department of Internal Medicine
I, University Hospital Frankfurt am Main, Theodor-Stern-Kai 7, D-60596
Frankfurt am Main, Germany. Fax: +49 69 6301 6405.
E-mail: [email protected]
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774 M. A. Pani et al.
2002 Blackwell Science Ltd, Clinical Endocrinology, 56, 773777
(DQ-LTR3) that is inserted only in some haplotypes (Donneret al.,
1999a). The presence of DQ-LTR3 enhances susceptibility to type
1 diabetes when linked to DR4-DQ8 (DRB1*04-DQB1*0302), or in
its absence on DQ2 (DQA1*0501-DQB1*0201) (Donneret al.,
1999b). A novel LTR 13 kb upstream of DQB1, denominated
DQ-LTR13 (Donner et al., 2000), is linked to distinct HLA
DQB1 haplotypes (Horton et al., 1998; Donneret al., 2000). We
found this LTR13 to confer additional susceptibility to type 1
diabetes (Bieda et al., 2002). Furthermore, a significant associ-
ation of DR4-DQ8-LTR3+
with rheumatoid arthritis was previously
reported by us (Seidl et al., 1999), and Pascual et al. (2001) who
also showed this for DQB1*0601-LTR3+.
Given the shared genetic susceptibility markers between type
1 diabetes and adrenal autoimmunity, we investigated the distri-
bution of DQ-LTR3 and DQ-LTR13 in patients with Addisons
disease compared to healthy controls.
Patients and methods
Subjects
Patients with Addisons disease (n = 87) were recruited at the
endocrine outpatient clinic at the University Hospital Frankfurt
am Main, Germany. Addisons disease was diagnosed by primary
adrenocortical insufficiency without evidence of tuberculosis or
adrenoleukodystrophy. 21-hydroxylase antibodies were found in
80% of patients (Seissler et al., 1999). Thirty-four patients
(358%) suffered either from thyroid autoimmune disease or were
thyroid autoantibody-positive as part of a polyglandular syn-drome type 2. None of the patients had type 1 diabetes. The age
of onset was 16 42 years and no neurological deficits could be
detected. Healthy controls (n = 160), who were randomly
collected from Frankfurt am Main, Germany, had no family
history of type 1 diabetes, Graves disease, Hashimotos thyroiditis
or Addisons disease. Although the controls adrenal function was
not formally assessed, all individuals had normal thyroid function
and were thyroid autoantibody-negative. Informed consent was
obtained from all individuals.
Genotype analysis
DNA was isolated from whole ethylenediaminetetraacetic acid
blood using standard protocols and subjected to polymerase
chain reaction (PCR) amplification. Each reaction included a
negative as well as a positive control. HLA-DQA1 and -DQB1
alleles were defined by sequence-specific primer analysis based
on the recent HLA nomenclature (Olerup et al., 1993; Marsh
et al., 2001) and typed as previously described (Badenhoop
et al., 1995).
The presence or absence of DQ-LTR3 and DQ-LTR13 was
defined by respective nested PCR approaches. Primer sequences
were deduced from the alignment of two previously published
HLA haplotypes (DQB1*0402 and *0201, GenBank #Z80898,
U92932) (Liao et al., 1998). For DQ-LTR3, external primers (5-
AAT GCT GAT TAG AAG TAG CTC TG-3 and 5-ACA AGG
ACA TCT CCT GAT CAG-3) were used to generate a 1285-bp
fragment in the presence of DQ-LTR3 or a 312-bp fragment in
its absence. The PCR program consisted of 30 cycles (94 C for
1 minute, 61 C for 1 minute, 72 C for 75 s) after 4 minutes initial
denaturation at 95 C and with a final extension of 4 minutes.
The internal DQ-LTR3 PCR was designed to generate a 1008-
bp fragment only in the presence of DQ-LTR3 using the oligo-
nucleotides 5-GGT GGA GCA ACA GCC CAC CCG GGA
AGT-3 and 5-CCC CTT GTG ACT TCT GTG GGG AAA
AGC-3 under the following conditions: initial denaturation
(94 C) for 4 minutes, 30 cycles (94 C for 1 minute, 62 C for
1 minute, 72 C for 1 minute) and a final extension of 4 minutes.
Detection of DQ-LTR13 was performed using external primers(5-GGT CAG AAG TAA TGT TTG CC-3 and 5-TAA TGG
TTA TAA AGC AAT TAG AAC-3) to generate a 1057-bp frag-
ment in the presence of DQ-LTR13 or a fragment of 51 bp in its
absence. The PCR program consisted of 30 cycles (94 C for
50 s, 57 C for 50 s, 72 C for 55 s) after 4 minutes initial dena-
turation at 95 C and with a final extension for 4 minutes. The
internal PCR of DQ-LTR13 was designed to generate a 1035-bp
fragment with primers 5-AGT AAT GTT TGC CAG TCT GTA
G-3 and 5-AAT TAG AAC AAT GCC TGG TGT G-3, located
to overlap the boundary of the noncoding HLA and DQ-LTR13
sequence. To verify these data obtained from the external and
internal PCR, we created a third PCR reaction with primers 5-CCAGTCTCAGGTGCTCTAGAA-3 and 5-AGAAGCATTT
CCTAGGTCCTGA-3 to generate a fragment of 1530 bp in the
presence of DQ-LTR13 or a 532-bp fragment in its absence. The
PCR program consisted of 30 cycles (94 C for 1 minute, 60 C
for 1 minute, 72 C for 1 minute 40 s) after 4 minutes initial
denaturation and with 4 minutes final extension.
All PCR fragments were separated on a 2% agarose gel and
stained with ethidium bromide (Roth, Karlsruhe, Germany). PCR
amplifications were carried out using Taq polymerase (Promega,
Madison, WI, USA) in the following reaction: 8 mM dNTPs,
15 pmol each primer, 10 mM Tris-HCl, 50 mM KCl, 15 mM
MgCl2 and 1 U Taq Polymerase. Whereas 200 ng genomic DNA
was used as template for external PCR, 2 l of a 1 : 100 dilution
of these amplification products was used for internal PCRs. All
amplifications were performed on a Multicycler PTC 200 (MJ
Research, Las Vegas, NV, USA).
Statistical analysis
Observed and expected genotype frequencies were compared
based on the HardyWeinberg equation. Patients and controls
were compared using allele-wise and genotype-wise chi-squared.
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HLA-DQ-LTR13 in Addisons disease 775
2002 Blackwell Science Ltd, Clinical Endocrinology, 56, 773777
All probabilities were corrected according to the respectivedegree of freedom (d.f.) andP< 005 was considered statistically
significant. The strength of association was estimated by the odds
ratio (OR) given with the respective 95% confidence interval
(CI). P-values were not corrected for the number of loci
(two: LTR3 and LTR13) investigated for an association with
Addisons disease. The power of each analysis is the likelihood
to reject a false null hypothesis (defined as 1 ). Power
calculations were performed using PASS 2000 (NCSS, Kaysville,
UT, USA).
Results
The observed DQ-LTR3 and DQ-LTR13 genotype frequencies
were in accordance to the HardyWeinberg equilibrium in both,
patients and controls (data not shown).
Subsequently, we compared the DQ-LTR3 and DQ-LTR13
distribution between patients and controls. DQ-LTR13+
geno-
types were significantly more frequent in patients: 368% of
patients carried the DQ-LTR13 insertion, but only 206% of
controls [genotype-wise chi-squared = 7587 (2 d.f.),P= 00225,
1 = 069]. Similarly, allele-wise analysis showed patients
alleles to carry DQ-LTR13 significantly more often than those
of controls (P= 00143, 1 = 069) (Table 1). In contrast, there
were no significant differences for DQ-LTR3 between patients
and controls both for genotype (P= 02103) and allele
(P= 00941) comparison (Table 2).
The HLA-DQ8 (DQA*0301-DQB*0302) haplotype was
significantly more frequent in patients than in controls [26 DQ8+
individuals of 87 (299%) patients vs. 24 (150%) DQ8+
out of
160 controls; chi-squared,P= 00089]. Because both DQ-LTR3
and DQ-LTR13 are in strong linkage disequilibrium with HLA-
DQ8, we analysed allele and genotype frequencies of DQ-LTR3
and DQ-LTR13 in DQ8+
patients and controls. Thereby, signi-
ficantly more DQ8+
patients carried the DQ-LTR13 insertion than
DQ8+
controls (genotype-wise chi-squared, P= 00008, 1
= 093) (Table 3). Again, no difference between patients and
controls was observed for the DQ-LTR3 insertion (Table 4).
No statistical analysis of the DQ2+
subset was possible due
to the absence of both LTR3 and LTR13 on the HLA-DQ2
haplotype.
Table 1 Distribution of DQ-LTR13 among all patients with Addisons
disease and all controls
Patients, n (%) Controls, n (%) OR (95% CI)
Genotype-wise comparison*
LTR13+/+ 1 (12) 1 (06) NS
LTR13+/ 31 (356) 32 (200) 221 (124395)
LTR13/ 55 (632) 127 (794) 045 (025079)
Allele-wise comparison
LTR13+ 33 (190) 34 (106) 197 (118329)
LTR13 141 (810) 286 (894) 051 (031085)
*Chi-squared = 7587 (2 d.f.): P= 00225; 1 = 069.
Chi-squared = 5996 (1 d.f.):P= 00143; 1 = 069.
OR, Odds ratio; CI, confidence interval.
Table 2 Distribution of DQ-LTR3 among all patients with Addisons
disease and all controls
Table 3 Distribution of DQ-LTR13 among DQ8+
patients with
Addisons disease and DQ8
+
controls
Table 4 Distribution of DQ-LTR3 among DQ8+
patients with Addisons
disease and DQ8+
controls
Patients, n (%) Controls, n (%) OR (95% CI)
Genotype-wise comparison*
LTR3+/+ 12 (138) 17 (106) NS
LTR3+/ 29 (333) 40 (250) NS
LTR3/ 46 (529) 103 (644) NS
Allele-wise comparison
LTR3+ 53 (305) 74 (231) NS
LTR3 121 (695) 246 (769) NS
*Chi-squared = 3119 (2 d.f.):P= 02103. Chi-squared = 2803 (1 d.f.):
P= 00941. OR, Odds ratio; CI, confidence interval.
Patients, n (%) Controls, n (%) OR (95% CI)
Genotype-wise comparison*
LTR13+/+ 1 (38) 1 (42) NS
LTR13+/ 21 (808) 7 (292) 1020 (2923567)
LTR13/ 4 (154) 16 (666) 009 (003033)
Allele-wise comparison
LTR13+ 23 (442) 9 (188) 344 (141838)
LTR13 29 (558) 39 (812) 029 (012071)
*Chi-squared = 14143 (2 d.f.):P= 00008; 1 = 093. Chi-squared
= 6323 (1 d.f.): P= 00119; 1 = 071. OR, Odds ratio; CI,
confidence interval.
Patients, n (%) Controls, n (%) OR (95% CI)
Genotype-wise comparison*
LTR3+/+ 8 (138) 7 (106) NS
LTR3+/ 15 (333) 14 (250) NS
LTR3/ 3 (529) 3 (644) NS
Allele-wise comparison
LTR3+ 31 (305) 28 (231) NS
LTR3 21 (695) 20 (769) NS
*Chi-squared = 0021 (2 d.f.):P= 09895. Chi-squared = 0005 (1 d.f.):
P= 09416. OR, Odds ratio; CI, confidence interval.
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2002 Blackwell Science Ltd, Clinical Endocrinology, 56, 773777
Discussion
In the present study, we investigated the role of DQ-LTR3 and
DQ-LTR13 in susceptibility to Addisons disease. Preliminary
evidence of DQ-LTR13, but not DQ-LTR3, being associated with
Addisons disease was found. As DQ-LTR3 and DQ-LTR13 were
in strong positive linkage disequilibrium with the HLA-DQ8
haplotype and DQ8 itself confers susceptibility to Addisons dis-
ease, we compared the occurrence of DQ-LTR3 and DQ-LTR13
in DQ8+
patients and controls. The association of the DQ-LTR13
insertion with susceptibility to Addisons disease was even
stronger in the DQ8+
subset, whereas no deviation from the
expected frequency was observed for DQ-LTR3. This suggests
the DQ-LTR13 insertion to confer susceptibility to Addisons
disease additional to HLA-DQ8 because, even after correction for
the number (n = 2) of loci tested, all differences remain significant.
Because Addisons disease is far less common than type 1diabetes or thyroid autoimmune disease, we could study only a
limited number of cases resulting in a power smaller than 08 in
some cases. Therefore, these data need to be confirmed in a larger
cohort of cases and controls, as well as in a family study. The
sample size would have to be increased by approximately one-
third to reach a power of 08 for all analyses investigating the
role of LTR13.
Due to the extensive linkage disequilibria within the HLA
region, our observation that LTR13 is associated with Addisons
disease may also be caused by an unknown locus that exerts a
primary effect on disease susceptibility. Although the parental
origin of HLA-DQ8 and of DQ-LTR13 cannot be unequivocallydetermined in a casecontrol study, there is strong evidence for
a selective linkage between DQ-LTR13 and HLA-DQ8. We pre-
viously found LTR13 to be present on more than 90% of DQ8
alleles, whereas it was predominantly absent (< 10% LTR13+
alleles) on most other HLA-DQ haplotypes, including DQ2
(Donner et al., 2000). Although we cannot exclude that DQ-
LTR13 may be inserted on the other haplotype in DQ8+
hetero-
zygous individuals, this is highly unlikely in the light of our
family data (Donneret al., 2000).
DQ-LTR13 was recently found to confer susceptibility to type
1 diabetes (Bieda et al., 2002) and to rheumatoid arthritis (Seidl
et al., unpublished data). DQ-LTR13 could have a role in the
pathogenesis of autoimmunity by influencing the transcription
of the adjacent DQB1 gene. LTR13 is situated 13 kb upstream
of the transcription start site of DQB1. Endogenous retroviral
elements are transcribed in various cell types, including
lymphocytes (Kambhu et al., 1990; Loweret al., 1993), and have
been detected in tissues from patients with Sjgrens syndrome
(Urnovitz & Murphy, 1996) and multiple sclerosis (Perron et al.,
1997). Functional studies on the promoter activity of LTR13 are
currently underway. Furthermore, endogenous retroviral
sequences encode superantigens capable of inducing a cell-
specific T cell response (Conradet al., 1997). Such superantigens
may be induced during a viral infection via interferon- induc-
tion (Staufferet al., 2001). Whether superantigen expression is
specific for cell autoimmune destruction or can also affect other
endocrine tissues is unknown at present (Loweret al., 1998).
In summary, this study provides preliminary evidence that
DQ-LTR13, but not DQ-LTR3, is associated with susceptibility
to Addisons disease. The fact that significantly more DQ8+
patients than DQ8+
controls carry the LTR13 insertion suggests
this endogenous retroviral element to be a risk marker in addition
to HLA-DQ. A family study would allow identification of the
parental origin of HLA-DQ8 and DQ-LTR13 haplotypes, as we
previously performed in type 1 diabetes (Bieda et al., 2002).
Preliminary data suggest LTR3/LTR13 act as enhancers for the
neighbouring DQB1 gene (Bieda et al., unpublished data).
Further functional investigations will help to improve our under-
standing of the regulatory properties of DQ-LTR13 and mighteventually reveal targets for immune intervention.
Acknowledgements
The study was funded by the Deutsche Forschungs gemein schaft
(DFG) grant Ba 976/8-1, 8-2.
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