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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


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



LTR3+/+ 12 (138) 17 (106) NS

LTR3+/ 29 (333) 40 (250) NS

LTR3/ 46 (529) 103 (644) NS


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.



LTR13+/+ 1 (38) 1 (42) NS

LTR13+/ 21 (808) 7 (292) 1020 (2923567)

LTR13/ 4 (154) 16 (666) 009 (003033)


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.



LTR3+/+ 8 (138) 7 (106) NS

LTR3+/ 15 (333) 14 (250) NS

LTR3/ 3 (529) 3 (644) NS


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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776 M. A. Pani et al.


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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