ORIGINAL
E n d o c r i n e
ARTICLE
R e s e a r c h
Dominant Suppression of Addison’s Disease
Associated with HLA-B15
Peter R. Baker, Erin E. Baschal, Pam R. Fain, Priyaanka Nanduri, Taylor M. Triolo,
Janet C. Siebert, Taylor K. Armstrong, Sunanda R. Babu, Marian J. Rewers,
Peter A. Gottlieb, Jennifer M. Barker, and George S. Eisenbarth
Barbara Davis Center for Childhood Diabetes (P.R.B., E.E.B., P.R.F., P.N., T.M.T., T.K.A., S.R.B., M.J.R.,
P.A.G., J.M.B., G.S.E.), University of Colorado Denver, Aurora, Colorado 80045-6511; and CytoAnalytics
(J.C.S.), Denver, Colorado 80209
Context: Autoimmune Addison’s disease (AD) is the major cause of primary adrenal failure in
developed nations. Autoantibodies to 21-hydroxylase (21OH-AA) are associated with increased risk
of progression to AD. Highest genetic risk is associated with the Major Histocompatibility region
(MHC), specifically human leukocyte antigen (HLA)-DR3 haplotypes (containing HLA-B8) and
HLA-DR4.
Objective: The objective of the study was the further characterization of AD risk associated with
MHC alleles.
Design, Setting, and Participants: MHC genotypes were determined for HLA-DRB1, DQA1, DQB1,
MICA, HLA-B, and HLA-A in 168 total individuals with 21OH-AA (85 with AD at referral and 83 with
positive 21OH-AA but without AD at referral).
Main Outcome Measure(s): Genotype was evaluated in 21OH-AA-positive individuals. Outcomes
were compared with general population controls and type 1 diabetes patients.
Results: In HLA-DR4⫹ individuals, HLA-B15 was found in only one of 55 (2%) with AD vs. 24 of 63
(40%) 21OH-AA-positive nonprogressors (P ⫽ 2 ⫻ 10⫺7) and 518 of 1558 (33%) HLA-DR4 patients
with type 1 diabetes (P ⫽ 1 ⫻ 10⫺8). On prospective follow-up, none of the HLA-B15-positive,
21-hydroxylase-positive individuals progressed to AD vs. 25% non-HLA-B15 autoantibody-positive
individuals by life table analysis (P ⫽ 0.03). Single nucleotide polymorphism analysis revealed the
HLA-DR/DQ region associated with risk and HLA-B15 were separated by multiple intervening single-nucleotide polymorphism haplotypes.
Conclusions: HLA-B15 is not associated with protection from 21OH-AA formation but is associated
with protection from progression to AD in 21OH-AA-positive individuals. To our knowledge, this
is one of the most dramatic examples of genetic disease suppression in individuals who already have
developed autoantibodies and of novel dominant suppression of an autoimmune disease by a class
I HLA allele. (J Clin Endocrinol Metab 96: 2154 –2162, 2011)
utoimmune Addison’s disease (AD) is an uncommon
disorder (1, 2) with insidious disease onset (e.g.
weight loss and fatigue) and acute manifestations (in the
form of Addisonian crisis) that can be fatal if not properly
A
recognized and treated. The formation of 21-hydroxylase
autoantibodies (21OH-AA) precedes the development of
AD in the absence of symptoms and is a marker for risk of
progression to clinical disease. This is similar to autoan-
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2011 by The Endocrine Society
doi: 10.1210/jc.2010-2964 Received December 20, 2010. Accepted April 19, 2011.
First Published Online May 11, 2011
Abbreviations: AD, Addison’s disease; APS-1, autoimmune polyendocrine syndrome, type
1; APS-2, autoimmune polyendocrine syndrome, type 2; DAISY, Diabetes Autoimmunity
Study of the Young; HLA, human leukocyte antigen; HLA-DR3, HLA-DRB1*0301DQB1*0201; HLA-DR4, HLA-DRB1*04-DQB1*0302; MHC, major histocompatibility complex; 21OH-AA, 21-hydroxylase autoantibodies; SNP, single-nucleotide polymorphism;
TAD, thyroid autoimmune disease; T1DGC, Type 1 Diabetes Genetics Consortium; T1DM,
type 1 diabetes mellitus.
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J Clin Endocrinol Metab, July 2011, 96(7):2154 –2162
tibody formation and disease progression in more common autoimmune diseases such as type 1 diabetes mellitus
(T1DM) and thyroiditis. More than 50% of individuals
with autoimmune AD have other autoimmune diseases
(including type 1 diabetes), making it likely that there is a
common pathophysiology (1, 3–5).
Autoimmune polyendocrine syndrome, type 2 (APS-2) often manifests as a combination of AD, T1DM, and/or autoimmune thyroid disease. In contrast to the monogenic autoimmune polyendocrine syndrome, type 1 (APS-1; in which
mutations occur in the AIRE gene), APS-2 is a polygenic
disorder with the primary susceptibility loci within the major
histocompatibility complex (MHC) (6 –12). Specific MHC
risk for AD (as an isolated disease or as part of APS-2) and
T1DM is associated with the class II MHC haplotypes human leukocyte antigen (HLA)-DRB1*0301-DQB1*0201
(HLA-DR3) and HLA-DRB1*04-DQB1*0302 (HLADR4) (1, 10). T1DM is more associated with DRB1*0401,
whereas AD is more associated with DRB1*0404 (1, 10, 13,
14) in the United States and Norway but not in Italian populations (15). Risk for AD and APS-2 has further been associated with an extended HLA-DR3 haplotype that includes a highly conserved MHC region from HLA-DR3 to
HLA-B8 (including the MICA5.1 allele) but less often includes HLA-A1 of the classic extended DR3-A1-B8 haplotype (8 –10, 16, 17).
Despite the known prevalence of extended HLA-DR4
haplotypes containing HLA-B15 in T1DM (6, 18), extended haplotypes of HLA-DR4 chromosomes (involving
class I alleles) have not been well defined for AD. Here we
describe a strikingly different class I association between
DR4 haplotypes in individuals with AD vs. individuals
with clinically isolated T1DM and individuals with
21OH-AA (many of whom also have T1DM) who have
not progressed to clinical AD. We find dominant suppression of progression to AD in 21OH-AA positive, nonAddisonian individuals who have HLA-B15.
Materials and Methods
Study design
In 1993 we began ongoing HLA genotype analysis of AD
referrals as well as 21OH-AA testing of relatives of AD individuals and individuals with T1DM. Follow-up for progression to
AD in 21OH-AA⫹ individuals continues through 2010. Subjects
with AD or 21OH-AA positivity were genotyped for HLA-B,
HLA-DR, and HLA-DQ. As illustrated in Fig. 1, we have enrolled a total of 168 individuals with positive 21OH-AA status.
There were 83 individuals with diagnosed AD upon referral, and
85 individuals without diagnosed AD but with positive
21OH-AA (including 11 who eventually were diagnosed with
AD after we detected positive 21OH-AA and 74 who have not
progressed). Subjects with 21OH-AA positivity were followed
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FIG. 1. Flow chart illustrating the number of 21OH-AA subjects
included in this study.
up from the date of first positive antibody detection until disease
onset or last follow-up without AD. Of those who progressed,
mean time to disease progression was 2.5 yr (confidence interval
95%, 1.5– 4.0 yr). Nonprogressors were followed up for a mean
of 5.2 yr (confidence interval 95%, 4.4 – 6.4 yr) from the time of
first antibody detection, with the absence of AD typically confirmed via clinical history, ACTH levels, and cortisol levels after
Cortrosyn stimulation.
Study population
The cohort with diagnosed AD has been previously described
(17). We have now studied 94 non-APS-1 individuals with AD.
There were 40 males and 54 females with the average age of AD
onset 26 yr (22 yr in males and 30 yr in females). Thirty-one of
94 individuals also had diabetes, and 36 had other autoimmune
diseases including thyroid disease, celiac disease, vitiligo, and
lupus. Thirty-nine individuals had no other reported autoimmune disease.
Acquisition of 21OH-AA⫹ individuals was through screening of T1DM patients (n ⫽ 67) or relatives of AD or T1DM
individuals (n ⫽ 16). Two 21OH-AA⫹ were referred for autoantibody testing on clinical suspicion of AD but did not progress
by clinical testing until more than a year after initial autoantibody positivity. Within the 21OH-AA-positive nonprogressor
cohort, 70 (80%) had type 1 diabetes and an additional six individuals had other autoimmune diseases (including autoimmune
thyroid disease, celiac disease, and vitiligo). Only 12 individuals
who were 21OH-AA positive had no other autoimmune disorders
reported; thus, the great majority would be classified as having
APS-2 (3). For those who volunteered race information (n ⫽ 126),
all were Caucasian [Hispanic (n ⫽ 4) and white non-Hispanic (n ⫽
122)]. Patients or their parents provided informed consent with
institutional review board oversight at the University of Colorado
Denver.
Inclusion criteria
AD patients were identified by several methods including referral with the diagnosis of AD from the National Adrenal Disease Foundation, referral of 21OH-AA-positive or symptomatic
relatives of Addison’s patients, and diabetic patients followed up
at the Barbara Davis Center for Childhood Diabetes diagnosed
with AD after 21OH-AA screening. All patients in this study
were 21OH-AA positive (index ⬎99th percentile of normal).
21OH-AA were measured with a fluid phase radioassay as
previously described (10). Patients with the clinical diagnosis
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of APS-1 (with distinct signs, symptoms, and abnormalities in
the AIRE gene) were excluded, and all included patients were
interferon-␣ autoantibody negative (confirming absence of
APS-1) (19).
Control data
HLA typing was available for families from the Type 1 Diabetes Genetics Consortium (T1DGC). The T1DGC enrolled
2300 affected sibling pairs with type 1 diabetes and their parents.
Completed typing for HLA alleles across the MHC region as well
as single-nucleotide polymorphism (SNP) typing more than
2800 SNPs across the MHC is available for all T1DGC individuals (20). Analyses in this paper used a single individual with
T1DM per family (n ⫽ 2300 individuals).
DR3/4 control individuals from the general population were
available from the Diabetes Autoimmunity Study of the Young
(DAISY) with HLA typing at birth of approximately 30,000
newborns. Details regarding the DAISY population are provided
in the paper from Rewers et al. 1996 (21). There were 271 HLADR3/4 (DRB1*0301-DQB1*0201/DRB1*04xx-DQB1*0302)positive, autoantibody-negative, nondiabetic, unrelated individuals with HLA-B and HLA-A allele typing available. HLA-B
frequency determination used only DR3/4-positive individuals,
given that this is the highest risk, most common genotype for AD
individuals.
Genotyping
We performed HLA-DRB1, HLA-DQB1, HLA-B, and
HLA-A typing using hybridization of linear arrays of immobilized, sequence-specific oligonucleotides with amplified exon 2
DNA similar to previously described methodology (22) and direct sequencing of amplified HLA-DRB1 exon 2 to differentiate
DRB1*04 subtypes.
MICA genotypes were determined using a fluorescent-based
method as reported previously (23–25). Briefly, PCR fragments
were generated using primers (MICA forward, 5⬘-/6-FAM/
CCTTTTTTTCAGGGAAAGTGC-3⬘; MICA reverse, 5⬘-CCTTACCATCTCCAGAAACTGC-3⬘) that flank the microsatellite
repeat polymorphism in the transmembrane region of the
MICA gene (exon 5). Reactions (25 ␮l) were assembled using
FailSafe PCR PreMix J, 2.5 U MasterAmp Taq polymerase
(Epicenter, Madison, WI), 10 nmol of each primer, and 100 ng
of genomic template. The PCR product was amplified via 35
PCR cycles of 94 C for 30 sec, 57 C for 35 sec, 72 C for 1 min,
and a final extension of 72 C for 45 min. Products were diluted
1:50 and were separated by capillary electrophoresis on an
ABI3100 Avant genetic analyzer (Applied Biosystems, Foster
City, CA). Alleles were identified using GeneMapper version 3.5
(Applied Biosystems). The approximate peak sizes corresponding to each allele are 180 for allele 4, 182.7 for allele 5, 184.1 for
allele 5.1, 185.7 for allele 6, and 194.5 for allele 9. The nomenclature used to define the MICA alleles was that of the MICA
sequences, 2000 (26).
SNP analysis
Two SNPs were selected to differentiate a common extended
DRB1*0404-DQB1*0302-B15 haplotype, with nearly identical
SNPs across the MHC, from all other DRB1*04-DQB1*0302B15 founder chromosomes with variable SNP patterns in the
T1DGC data set based on previously described methods (27).
The SNPs (rs2242657 C and rs2269475 T) were analyzed in the
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DR4-B15⫹, 21OH-AA⫹, nonprogressor population (n ⫽ 19
with usable DNA), available family members, and selected
T1DGC controls using Taqman probes (hybridization/extension
reaction with a fluorescence detection system developed by Applied Biosystems). To unambiguously assign phase for sentinel
SNP typing in 21OH-AA positive nonprogressors, we analyzed
family data as well as used homozygosity of the sentinel SNPs.
Statistical analysis
The Fisher’s exact test (two sided) was used to calculate P
values for association with AD, with ␣ ⫽ 0.05. PRISM GraphPad
4 software (GraphPad Inc., San Diego, CA) was used for ␹2 (␣ ⫽
0.05). Life tables were also generated using PRISM GraphPad 4
software, wherein time to progression to AD (or last disease free
clinical follow-up) from first detected 21OH-AA were placed in
a nonlinear regression model comparing presence or absence of
HLA-B15 in various subgroups.
Results
We tested 21OH-AA in individuals referred with AD (AD
referrals), the relatives of AD patients, and patients with
T1DM only and identified a total of 168 21OH-AA⫹
individuals (Fig. 1). Autoantibodies to 21-hydroxylase are
typically present in 1.5% of non-Addisonian patients with
type 1 diabetes and in more than 90% of patients with
autoimmune AD (1, 10). The great majority of non-AD,
21OH-AA⫹ individuals enrolled in the current study had
T1DM (80%, n ⫽ 68 of 85) as did 33% of the referred
patients with existing AD (n ⫽ 31 of 94).
To fix HLA class II genotypes and analyze HLA-B allele
association with AD, we initially analyzed DRB1*0301DQB1*0201/DRB1*04-DQB1*0302 (DR3/4) individuals (the highest risk MHC class II genotype for AD). As
illustrated in Fig. 2, in DR3/4 individuals HLA-B15 was
the only HLA-B allele significantly decreased in AD
(DR3/4 AD ⫽ 0 of 43, DR3/4 general population controls ⫽ 58 of 271, P ⫽ 1.7 ⫻ 10⫺4, odds ratio 0.04). Of
note, HLA-B15 is rarely on HLA-DR3 haplotypes but frequently (and most commonly), it is associated with HLADR4 haplotypes (18). As we have reported previously, the
HLA-B8 allele is positively associated with AD in DR3positive individuals (not shown in the figure) and was
present in 91% (39 of 43) of DR3/4 patients with AD vs.
64% (174 of 271) of DR3/4 DAISY general population
controls (P ⫽ 3 ⫻ 10⫺4).
This negative association of HLA-B15 with AD is not
limited to DR3/4 subjects. As shown in Fig. 3A, for all
AD subjects (n ⫽ 94), only 4% had HLA-B15 (including
progressors who were followed up to AD and patients
with existing AD at referral) compared with 36% of
21OH-AA positive nonprogressors (27 of 74, P ⫽ 1 ⫻
10⫺7) and 25% of patients with T1DM (564 of 2213,
P ⫽ 1 ⫻ 10⫺7). After stratifying for HLA-DR4⫹ indi-
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of 63) of 21OH-AA⫹ nonprogressors
(P ⫽ 0.05). Comparison of HLA-DR4
positive nonprogressors and individuals with T1DM reveals no significant difference in HLA-B15 frequency [24 of 63 (40%) and 518 of
1558 (34%), respectively, P ⫽ 0.34],
indicating that HLA-B15 is not associated with decreased development of
21OH-AA. Among non-DR4 patients
with 21OH-AA positivity, relatively
few individuals had HLA-B15 including three of 39 Addison’s patients
(8%) compared with three of 12 nonprogressors (25%) (P ⫽ 0.13, not
significant).
We prospectively follow up individuFIG. 2. HLA-DR3/4 AD patients and general population (DAISY) controls were evaluated for
als who express 21OH-AA for progresHLA-B allele frequency. Within each bar the number indicates the number of individuals with
sion to AD (Fig. 1). Despite expressing
the given HLA-B allele. HLA-B15 was the only HLA-B allele to be significantly decreased in the
21OH-AA, none of the HLA-B15-posi⫺3
AD subjects. Corrected P value for HLA-B15 positivity in HLA-DR3/4 is 2.3 ⫻ 10 .
tive individuals have progressed to AD
with up to 13 yr of follow-up. In contrast,
viduals in these three cohorts (Fig. 3B), the HLA-B15
allele is almost entirely absent in HLA-DR4⫹ individ- nearly 25% of all non-HLA-B15, 21OH-AA-positive indiuals with AD [one of 55 (2%)], but it is common in viduals progressed by 13 yr of follow-up by life table analysis
DR4⫹, 21OH-AA-positive nonprogressors [24 of 63 (Fig. 4A, P ⫽ 0.03). Just analyzing the highest-risk HLA
genotype (DR3/4 positive, HLA-B8 positive individuals),
(40%), P ⫽ 2 ⫻ 10⫺7].
With the known association of HLA-DRB1*0404 in 75% of those lacking HLA-B15 progressed to AD by 7 yr of
AD and HLA-DRB1*0401 in T1DM as well as the high follow-up compared with 0% of those with HLA-B15 (P ⫽
prevalence of T1DM in our 21OH-AA⫹ nonprogressor 0.03).
To better define HLA-DR4-B15 extended haplotypes, we
cohort, we examined the frequency of HLA-DRB1*0404
in 21OH-AA⫹ nonprogressors vs. those with AD. analyzed SNPs previously typed across the MHC by the
DRB1*0404 in 21OH-AA positive nonprogressors (n ⫽ T1DGC consortium. Figure 5 illustrates SNPs across the
33 of 63, 52%) and Addison’s disease individuals (n ⫽ 39 MHC region in DRB1*0401-B15 and DRB1*0404-B15
of 56, 67%) were increased relative to patients with founder chromosomes analyzing the large T1DGC diabetes
T1DM [275 of 1605 (17%), P ⫽ 3.7 ⫻ 10⫺17 and 5.7 ⫻ family study (Fig. 5A). A conserved extended DRB1*040110⫺10, respectively]. Likewise, DRB1*0401 was present B15 haplotype was present in approximately two thirds (294
in 20% (11 of 56) of individuals with AD vs. 36% (23 of 446) of the DRB1*0401-B15 chromosomes (all yellow).
FIG. 3. HLA-DRB1, HLA-DQB1, and HLA-B were evaluated in AD individuals (including 21-hydroxylase positive progressors), 21-hydroxylasepositive individuals who have not progressed to AD [21-OH⫹(NP)], and patients with T1DM (DM). A, Percent HLA-B15 in all subjects genotyped for
the aforementioned loci. B. Percent HLA-B15 in only the HLA-DR4 positive subjects. All P values listed are relative to AD.
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FIG. 4. Life table analysis showing protection associated with HLA-B15 in 21OH-AA-positive individuals. A, All genotyped subjects. B, Only
DR3-B8/DR4 (the highest risk genotype) subjects.
The conservation of the SNP haplotype begins to break up
between HLA-B and HLA-A (with alternative SNPs colored in blue). Among potential variable SNPs, we chose
two (rs2242657 and rs2269475) to mark haplotypic variation in the region between HLA-DRB1 and HLA-B in
DR4-B15 haplotypes (represented as the 2-SNP haplotypes CT to mark the common DRB1*0404-like conserved haplotype or TC to mark alternative haplotypes).
As shown in Fig. 5B, the DR4-B15 haplotypes of 21OHAA-positive nonprogressors show variation between
DRB1 and HLA-B and therefore did not show evidence of
a single conserved, long-range MHC haplotype associated
with lack of progression.
Discussion
For polygenic autoimmune diseases such as T1DM and
AD, the MHC region on chromosome 6 is the most
important determinant of disease risk (7–10). Within
the MHC region, alleles of class II MHC molecules such
as DR and DQ are most strongly associated with AD
and APS-2 (1, 10, 13, 14, 16, 17). These are immune
response genes whose function is to present peptides to
T cell receptors of CD4⫹ T cells. Conversely, the class I
MHC molecules (e.g. HLA-B) classically present peptides to
cytotoxic T cells (CD8⫹). An example of HLA class II
influence on autoimmune disease is the genotype conferring high risk for AD and T1DM: DR3/4 (DRB1*0301DQB1*0201/DRB1*04xx-DQB1*0302) (1, 10, 13, 14). In
addition to class II HLA alleles, select class I alleles have
been reported to increase risk of type 1 diabetes (e.g.
HLA-B3906 and HLA-A24) (28 –31) and AD (16, 17).
Although the HLA-DR3 haplotype association with
AD has been well defined, with the highest risk determined by the presence of HLA-B8 on a conserved HLADR3 haplotype often without HLA-A1 (17), there are
fewer data regarding haplotypes of chromosomes with
HLA-DR4.
We have found that HLA-B15 is associated with dramatic protection from progression to AD in DR4-positive individuals, even in individuals who have already
developed 21OH-AA. This is the first report to our
knowledge of a class I HLA allele associated with dominant suppression of autoimmune disease in individuals
who have already developed autoantibodies. Looking
at individuals who have already developed 21OH-AA
but who have not progressed to AD, a total of 27 of 74
have HLA-B15, in contrast to patients with AD (four of
94, P ⫽ 6 ⫻ 10⫺8, odds ratio 0.07). By life table analysis,
risk of progression to AD is greatly diminished in the
presence of HLA-B15 (see Fig. 4). When specifically
looking at the high-risk genotype DR3/4, none of the
Addison’s patients had the HLA-B15 allele (n ⫽ none of
43), whereas it was present in 35% of 21OH-AA-positive DR3/4 nonprogressors (n ⫽ 13 of 37, P ⫽ 1 ⫻
10⫺5), 29% of DR3/4 individuals with T1DM (n ⫽ 261
of 910, P ⫽ 1.4 ⫻ 10⫺6) and 21% of DR3/4 controls
(n ⫽ 58 of 271, P ⫽ 2 ⫻ 10⫺4).
Given the heterogeneity of autoimmune AD presentation,
specifically when it is either isolated or as a part of APS-2
[with T1DM, thyroid autoimmune disease (TAD), or both],
it is important to ensure the effect seen with HLA-B15 is not
due to association with another comorbid disorder. When
looking at the AD and 21OH-AA⫹ nonprogressor individuals with known comorbidity status, we found that there is
still a significant difference in HLA-B15 presence between
the two groups when comorbidities are accounted for. In
isolated 21OH-AA positivity (without either T1DM or
TAD), 30% (three of 10) of individuals had HLA-B15 vs. 5%
(two of 39) in the AD group (P ⫽ 0.05). Similarly, among
those with AD or 21OH-AA nonprogressors with T1DM,
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FIG. 5. HLA-DR-DQ and HLA-B regions of conservation are noncontiguous (separated by polymorphic regions). A, SNPs across the MHC region.
Each column represents a single DR4-B15 founder chromosome (n ⫽ 556) in the Type 1 Diabetes Genetics Consortium database of families with
phased defined SNP haplotypes. Each row represents a SNP (n ⫽ 2828) with the allele in yellow if identical to the allele on the most common
conserved DR401-B15 haplotype, in blue if the opposite allele, and in white if the allele could not be defined. The white column separates
DRB1*0401 from DRB1*0404 T1DGC chromosomes. B, HLA genotype and sentinel SNP analysis in DR4-B15 chromosomes of 21OH-AA-positive
nonprogressors. Alleles similar to the most common conserved DR404-B15 haplotype are seen in green, whereas alleles commonly on DR401-B15
haplotypes are in yellow. Orange represents non-401, non-404 haplotypes. Gene positions (indicated in parentheses in A) are based on National
Center for Biotechnology Information Human Genome build 36.
TAD, or both, 38% (24 of 63) of nonprogressors vs. 4% (two
of 53) of AD had HLA-B15 (P ⫽ 4.5 ⫻ 10⫺6).
Because alleles of genes in the MHC are often in
long-range linkage disequilibrium (spanning millions of
base pairs) (6), it is difficult to determine whether the
effect of HLA-B15 is from the specific HLA-B15 allele
or from the haplotypes with which it is associated. With
known protective effects of DRB1-DQB1 genotypes
(including those with DRB1*0403), it is important to
assess HLA-B15 associated protection in the context of
class II alleles. HLA-B15 is not typically found with
DRB1*0403. In the 21OH-AA⫹ nonprogressor group,
only one of 27 individuals with HLA-B15 (4%) also had
DRB1*0403 vs. only two of 560 (⬍1%, P ⫽ 0.13) of
HLA-B15⫹T1DGC individuals and none of four AD
individuals (P ⫽ 1). Looking at our DR3/4 general population controls, only three of 23 individuals with
DRB1*0403 had HLA-B15.
Further analysis of sentinel SNPs in the predominantly
DRB1*0404 and DRB1*0401 chromosomes indicates that
HLA-DR4-B15 haplotypes are polymorphic between DRB1
and HLA-B15. Although long-range association with variation in between these two loci is a formal possibility, our
findings suggest that at least two distinct MHC regions contribute to AD (class II and class I), with the simplest hypothesis positing direct influence of HLA-B15 on AD risk.
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The paucity of HLA-B15 in AD populations also leads
to an interesting observation regarding MICA 5.1, an allele reported to be associated with AD risk (10). MICA is
located physically close to HLA-B and is associated with
the DR3-B8 haplotype, and the MICA 5.1 allele (which is
in strong linkage with HLA-B8) has been associated with
increased risk of AD (7, 17, 24). Specifically we reported
MICA5.1 homozygosity to be associated with an increased risk of progression to AD in 21OH-AA-positive
subjects (10). Part of this association may be explained by
MICA5.1’s inclusion in the DR3-B8 extended haplotype,
but given that DR3-B8 homozygosity is relatively uncommon in AD individuals (six of 94, 6%), increased homozygosity of MICA5.1 (48 of 94, 51%) in AD indicates nonDR3-B8 haplotypes with MICA5.1 is associated risk. Of
note, there is strong linkage disequilibrium between HLAB15 and MICA 5 (not MICA 5.1), which has been independently associated with other immune mediated disorders
(e.g. giant cell arteritis and aggressive forms of periodontal
disease) (32, 33).
In our series of DR3-B8/DR4 individuals, MICA 5.1/
5.1 is found in 28% of 21OH-AA nonprogressors (n ⫽ six
of 21) vs. 62% of Addison’s individuals (n ⫽ 24 of 39, P ⫽
0.01). However, when the data are stratified for HLA-B15
(thus removing HLA-B15 positive individuals), almost
50% of 21OH-AA nonprogressors have MICA5.1/5.1,
making the difference of MICA 5.1 homozygosity between this group and AD group nonsignificant. Thus, we
theorize that the MICA 5.1 homozygosity association
with AD may be secondary to linkage disequilibrium of
MICA 5.1 with high-risk HLA-B8 haplotypes as well as by
the MICA 5 linkage disequilibrium with HLA-B15 lowrisk haplotypes associated with decreased risk. To directly
test this hypothesis, analysis of larger series and potentially other ethnic groups (with different patterns of linkage disequilibrium) will be needed.
If HLA-B15 itself is protective, its mechanism of action
would be of interest because AD is primarily associated
with class II HLA alleles. Increased disease risk with class
I HLA alleles is typically ascribed to enhanced presentation of autoantigenic peptides to CD8⫹ T cells. There are
reports of class I HLA alleles negatively associated with
autoimmune disease (34) along with literature supporting
the existence of CD8⫹ regulatory T cells, which are HLA
class I restricted (35–38). This mechanism of pathogenesis, and its possible relation to HLA-related autoimmune
disease, warrants further exploration.
Protection mediated by the molecule HLA-B15 likely
involves MHC class I structure and the polymorphic
amino acids that line the binding pocket. When looking at
amino acid sequences in HLA-B*0801 (associated with
highest AD risk) and HLA-B*1501, there are 23 total
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amino acids that differ including seven changes in the
binding pocket base and five in the ␣-helix of the binding
groove (39, 40). Sequence differences of multiple alleles
will need to be further explored using molecular modeling,
pending analysis of larger numbers and multiple populations of individuals with AD.
Even with 21OH-AA and the DR3-B8/DRB1*0404
highest-risk genotype, HLA-B15 (or linked loci) protects
from progression to overt AD. This study illustrates the
potential of analysis of HLA haplotypes in determining
risk in antibody-positive populations. We typically screen
patients with T1DM for 21OH-AA, and we annually evaluate adrenal function for those found to be positive. Although AD has classic symptoms and signs, in patients
with T1DM initial presentation can be as subtle as increased occurrences of hypoglycemia and decreasing insulin requirement (without hyperpigmentation). If further prospective studies support our findings, the
approximate one third of 21OH-AA-positive patients
who also have HLA-B15 are at greatly decreased risk of
progression to AD.
Acknowledgments
Address all correspondence and requests for reprints to: George
S. Eisenbarth, Barbara Davis Center for Childhood Diabetes,
1775 Aurora Court, Room 4201E, Building M20, P.O. Box
6511, Box B140, Aurora, Colorado 80045-6511. E-mail:
[email protected].
This work was supported by the Type 1 Diabetes Genetics Consortium, a collaborative clinical study sponsored by the National
Institute of Diabetes and Digestive and Kidney Diseases, National
Institute of Allergy and Infectious Diseases, National Human Genome Research Institute, the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and Juvenile Diabetes Research Foundation International and supported by Grant
U01 DK062418. This work was also supported by the National
Institutes of Health Grant DK32083, Diabetes Autoimmunity
Study in the Young Grant DK32493, Autoimmunity Prevention
Center Grant AI050864, Diabetes Endocrine Research Center
Grant P30 DK57516, Clinical Research Centers Grants MO1
RR00069 and MO1 RR00051, the Immune Tolerance Network
Grant AI15416, the American Diabetes Association, the Juvenile
Diabetes Research Foundation Grant 11-2005-15, the Children’s
Diabetes Foundation, and the Brehm Coalition. P.R.B. is a Fellow
of the Pediatric Scientist Development Program. The project described was also supported by Award K12-HD000850 from the
Eunice Kennedy Shriver National Institute of Child Health and
Human Development.
Disclosure Summary: The authors have no conflicts of interest to disclose.
J Clin Endocrinol Metab, July 2011, 96(7):2154 –2162
jcem.endojournals.org
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