Effect of HLA class I and class II alleles on progression from autoantibody positivity to overt type 1 diabetes in children with risk-associated class II genotypes.

Abstract

Class II alleles define the main HLA effect on type 1 diabetes, but there is an independent effect of certain class I alleles. Class II and class I molecules are differently involved in the initiation and effector phases of the immune response, suggesting that class I alleles would be important determinants in the rate of β-cell destruction. To test this hypothesis we analyzed the role of HLA class I and class II gene polymorphisms in the progression from diabetes-associated autoimmunity to clinical disease.
The effect of HLA-DR-DQ haplotypes and a panel of class I HLA-A and -B alleles on the progression from autoantibody seroconversion to clinical diabetes was studied in 249 children persistently positive for at least one biochemical diabetes-associated autoantibody in addition to islet cell autoantibody.
The progression to clinical disease was separately analyzed after the appearance of the first and the second persistent biochemical autoantibody using Cox regression. Multivariate analysis demonstrated a significant protective effect of the A*03 allele (odds ratio [OR] 0.61, P = 0.042 after the first and OR 0.55, P = 0.027 after the second autoantibody), whereas the B*39 allele had a promoting effect after seroconversion for the second autoantibody (OR 2.4, P = 0.014). When children with the DR3/DR4 genotype were separately analyzed, HLA-B*39 had a strong effect (OR 6.6, P = 0.004 and OR 7.5, P = 0.007, after the appearance of the first and the second autoantibody, respectively). The protective effect of A*03 was seen only among children without the DR3/DR4 combination.
These results confirm that class I alleles affect the progression of diabetes-associated autoimmunity and demonstrate interactions between class I and class II alleles.

Full text

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Effect of HLA Class I and Class II Alleles on Progression
From Autoantibody Positivity to Overt Type 1 Diabetes in
Children With Risk-Associated Class II Genotypes
Kati Lipponen,1Zsofia Gombos,1Minna Kiviniemi,1Heli Siljander,2,3Johanna Lempainen,1
Robert Hermann,1Riitta Veijola,4Olli Simell,5Mikael Knip,2,3and Jorma Ilonen1,6
OBJECTIVE—Class II alleles define the main HLA effect on type
1 diabetes, but there is an independent effect of certain class I
alleles. Class II and class I molecules are differently involved in
the initiation and effector phases of the immune response,
suggesting that class I alleles would be important determinants in
the rate of ?-cell destruction. To test this hypothesis we analyzed
the role of HLA class I and class II gene polymorphisms in the
progression from diabetes-associated autoimmunity to clinical
disease.
RESEARCH DESIGN AND METHODS—The effect of HLA-
DR-DQ haplotypes and a panel of class I HLA-A and -B alleles on
the progression from autoantibody seroconversion to clinical
diabetes was studied in 249 children persistently positive for at
least one biochemical diabetes-associated autoantibody in addi-
tion to islet cell autoantibody.
RESULTS—The progression to clinical disease was separately
analyzed after the appearance of the first and the second
persistent biochemical autoantibody using Cox regression. Mul-
tivariate analysis demonstrated a significant protective effect of
the A*03 allele (odds ratio [OR] 0.61, P ? 0.042 after the first and
OR 0.55, P ? 0.027 after the second autoantibody), whereas the
B*39 allele had a promoting effect after seroconversion for the
second autoantibody (OR 2.4, P ? 0.014). When children with
the DR3/DR4 genotype were separately analyzed, HLA-B*39 had
a strong effect (OR 6.6, P ? 0.004 and OR 7.5, P ? 0.007, after the
appearance of the first and the second autoantibody, respec-
tively). The protective effect of A*03 was seen only among
children without the DR3/DR4 combination.
CONCLUSIONS—These results confirm that class I alleles
affect the progression of diabetes-associated autoimmunity and
demonstrate interactions between class I and class II alleles.
Diabetes 59:3253–3256, 2010
T
circulating autoantibodies as a marker of the ongoing
autoimmune process, and the duration of this period is
highly variable (1). There may be differences in the genetic
and environmental factors affecting the initiation of the
autoimmune response and the later course of ?-cell auto-
immunity, conceivably leading to clinical disease. This has
also been suspected for genes within the HLA region
where susceptibility to type 1 diabetes is mainly defined by
alleles of class II DR and DQ genes, although evidence has
also accumulated for the contribution of class I alleles in
the A and B loci (2–5). It has been proposed that class II
genes determine the initiation of autoimmunity, whereas
class I genes define the progression of ?-cell damage (6).
This model is theoretically supported by the roles of class
II and class I molecules in the immune response. This
response is initiated when CD4? T-cells recognize anti-
gens in the context of class II HLA molecules, whereas
cytotoxic CD8? T-cells respond to antigenic peptides
presented by the class I molecules.
To further explore this hypothesis we analyzed the
effect of common class II haplotypes and a panel of class
I alleles on the rate of progression to clinical diabetes in a
follow-up group of children with established diabetes-
associated autoimmunity. This cohort of 249 children was
derived from the Finnish Diabetes Prediction and Preven-
tion (DIPP) study. All the subjects had at least one
persistently positive biochemically defined autoantibody
in addition to islet cell autoantibodies (ICAs); of these, 136
(54.6%) developed type 1 diabetes during the follow-up
period.
he HLA gene region on the short arm of chromo-
some 6 is the most important of the multiple
gene loci affecting susceptibility to type 1 diabe-
tes. Disease onset is preceded by the presence of
RESEARCH DESIGN AND METHODS
The newborn infants were recruited to the DIPP study in three university
hospitals in Finland: Turku, Oulu, and Tampere. After initial screening for
HLA-DQ–associated genetic risk, the follow-up group was sampled at 3- to
12-month intervals and serum tested for ICA. Originally, newborns positive for
HLA-DQB1*0302 and without DQB1*0301 or DQB1*0602/3 alleles were se-
lected for the study, but later those with DQB1*0302/DQB1*0603 and boys
with DQA1*05-DQB1*02 without DQA1*0201, DQB1*0301, or DQB1*0602/3
were also accepted (7). If ICAs were found to be positive, all samples available
from that individual were tested for biochemically defined autoantibodies, i.e.,
insulin autoantibodies (IAA), and antibodies to the 65 kDa isoform of GAD
(GADA) and to the protein tyrosine phosphatase related IA-2 molecule
(IA-2A). All ICA-positive children who tested persistently positive (at least two
consecutive positive samples taken at an interval of 3 months or longer) for at
least one biochemically defined autoantibody besides ICA and whose sample
was available for further genotyping (N ? 249) were selected for the study. Of
them, 195 were persistently positive for two or three biochemically defined
From the1Immunogenetics Laboratory, University of Turku, Turku, Finland;
the
Helsinki University Central Hospital, Helsinki, Finland; the3Department of
Pediatrics, Tampere University Hospital, Tampere, Finland; the
ment of Pediatrics, University of Oulu, Oulu, Finland; the5Department of
Pediatrics, University of Turku, Turku, Finland; and the
Clinical Microbiology, University of Eastern Finland, Kuopio, Finland.
Corresponding author: Jorma Ilonen, jorma.ilonen@utu.fi.
Received 3 February 2010 and accepted 9 August 2010. Published ahead of
print at http://diabetes.diabetesjournals.org on 25 August 2010. DOI:
10.2337/db10-0167.
© 2010 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
2Hospital for Children and Adolescents, University of Helsinki and
4Depart-
6Department of
BRIEF REPORT
diabetes.diabetesjournals.orgDIABETES, VOL. 59, DECEMBER 20103253

Page 2

autoantibodies. Altogether, 201 of the children (80.7%) tested positive for IAA,
184 (73.1%) for GADA, and 176 (70.6%) for IA-2A. The lack of persistent
autoantibody positivity was accepted only when the autoantibody was de-
tected for the first time at the time of the diagnosis of diabetes because no
further samples were available after the diagnosis.
The median age of the children at the time of seroconversion to positivity
for the first biochemically defined autoantibody was 1.8 years (range 0.3–9.9)
and 2.0 years (0.8–9.9) for the second biochemically defined autoantibody.
Median follow-up time from the first biochemically defined autoantibody was
6.4 years (0.5–12.5) in the 113 children remaining nondiabetic and 2.8 years
(0.0–10.8) in the 136 children progressing to type 1 diabetes. Median follow-up
time from the appearance of the second biochemically defined autoantibody
was 6.1 years (1.8–12.0) in the 78 children remaining unaffected and 2.5 years
(0.0–5.8 years) in the 117 children progressing to type 1 diabetes. The
diagnosis of type 1 diabetes was based on the World Health Organization
criteria (8).
Genotyping methods. HLA class II typing was performed as described
earlier (9,10) using a panel of lanthanide-labeled oligonucleotide probes.
HLA-DQB1 alleles were analyzed as the first step and -DQA1 and -DRB1 alleles
thereafter as needed for the haplotype deduction. The typing protocol defined
the presence of common European HLA-DR-DQ haplotypes with special
reference to those associated with diabetes risk (11). The high-risk genotypes
formed by HLA-(DR3)-DQA1*05-DQB1*02 and HLA-DRB1*0401-DQB1*0302 or
HLA-DRB1*0404-DQB1*0302 haplotypes were combined and named the DR3/
DR4 genotype. All 249 children were successfully typed for the presence of
class II haplotypes. The common HLA-A and -B alleles in the Finnish
population were typed using allele-specific amplification and detection of
amplification products on agarose gels (12). The defined alleles include those
commonly detected in type 1 diabetes–associated HLA-DR3 and -DR4 positive
haplotypes (5,13). The presence of HLA-A*01, -A*02, -A*03, -A*24, -A*28,
-A*32, -B*08, -B*27, -B*35, -B*39, -B*56, -B*60, and -B*62 alleles could be
defined in most cases, but the typing did not give a definite result in a subset
of samples varying from 38 (15.3%) for -A*32 to 57 (22.1%) for -B*56.
Statistical analysis. The effect of various HLA alleles, haplotypes, and
genotypes on the progression to clinical disease was tested applying Cox
regression analysis. The PASW 18.0 statistical software (SPSS Inc., Chicago,
IL) was used for the analyses.
RESULTS
We started by using the Cox regression univariate analysis
to test the effect of all typed class I alleles and class II
haplotypes on the progression to type 1 diabetes after the
appearance of the first biochemically defined autoantibody
as well as after the appearance of at least two biochemi-
cally defined autoantibodies. Those class I alleles with a
significant effect (P ? 0.05) in the analysis carried out after
the appearance of either one or two persistently positive
biochemical autoantibodies were selected for multivariate
analysis together with the HLA-DR3/DR4 genotype. This
genotype showed a significant association in the univariate
analysis (P ? 0.010) with progress to disease when tested
after the appearance of the first biochemical autoantibody.
When the effect of the HLA-DR-DQ haplotypes was tested,
(DR3)-DQA1*05-DQB1*02 was found to be associated with
progression to type 1 diabetes (P ? 0.015) and (DR1/10)-
DQB1*0501 with protection against overt disease (P ?
0.03). Similar associations were also found when these
twohaplotypeswere associated
DQB1*0302; these haplotype associations were most prob-
ably secondary to the DR3/DR4 genotype association
because of the selection criteria used for the majority of
the study participants. (DR3)-DQA1*05-DQB1*02 was
found in the DR3/DR4 genotype in 80 of 88 (90.9%)
participants, whereas (DR1/10)-DQB1*0501 is the major
“neutral” haplotype in the Finnish population, thus repre-
senting those children lacking DR3/DR4.
Table 1 shows the result of the multivariate analyses
including HLA-A*03, *A24 and *B39 alleles and the DR3/
DR4 genotype adjusted for the age at seroconversion for
the first or second biochemical autoantibody. HLA-A*03
was associated with protection against disease in both
withDRB1*0401-
analyses but HLA-B*39 with progression of autoimmunity
only in the analysis after the appearance of the second
autoantibody. HLA-DR3/DR4 had a tendency to associate
with progressing autoimmunity after the appearance of the
first autoantibody, but no effect was detected in the
analysis after seroconversion for two autoantibodies.
We also performed the same analysis with class I alleles
after dividing the children according to the presence of the
HLA-DR3/DR4 genotype (Table 2). This revealed a strong
association of HLA-B*39 with progression to diabetes
when associated with the HLA-DR3/DR4 genotype, but not
in children with other class II genotypes. The rate of
disease development is also shown in the Kaplan-Meier
curve in Fig. 1, and the number of children in follow-up at
each time point is also given.
DISCUSSION
Our results confirm the strong effect of two class I alleles
on the progression of type 1 diabetes–associated autoim-
munity. The possibility that other linked genes in the
major histocompatibility complex block are behind these
findings cannot be excluded, but the binding of autoim-
mune epitopes efficiently presented to cytotoxic CD8 cells
by these class I alleles is an attractive hypothesis for the
operative mechanism.
Although it is clearly demonstrated that class II HLA
genotypes associated with type 1 diabetes are also linked
to positivity for multiple autoantibodies predicting the
development of clinical disease (7,14,15), their additional
role in affecting the progression of ?-cell autoimmunity
cannot be excluded. Two studies based on the Diabetes
Prevention Trial–Type 1 (DPT-1) series have set out to
analyze the effect of class II HLA alleles on the progression
to type 1 diabetes among autoantibody-positive first-de-
gree relatives. Redondo et al. (16) concluded that the
strong class II genotype effects observed were mainly due
to differences in the initial autoantibody profiles of the
study subjects. The other DPT-1 study selected only sub-
jects with ICA and IAA with initially preserved ?-cell
function and reported that the DQB1*0302 allele was
associated with progression while DQB1*0301 was related
to protection against type 1 diabetes (17). The DPT-1
TABLE 1
Cox regression analysis of HLA effects on progression to clinical
diabetes in children with diabetes-associated autoantibodies
Follow-up from appearance of first biochemically defined
autoantibody*
Multivariate analysis
HLA-A*03
HLA-A*24
HLA-B*39
HLA-DR3/4
0.042
0.191
0.159
0.070
0.611 (0.380–0.982)
1.422 (0.839–2.411)
1.549 (0.843–2.844)
1.482 (0.968–2.269)
Follow-up from appearance of second biochemically defined
autoantibody†
HLA-A*030.027
HLA-A*240.598
HLA-B*390.014
HLA-DR3/40.809
0.552 (0.326–0.933)
1.176 (0.644–2.146)
2.401 (1.196–4.823)
1.063 (0.647–1.746)
Data are P values and hazard ratio for diabetes progression (95% CI).
*Stratified according to age at appearance of first biochemically defined
autoantibody. †Stratified according to age at appearance of second
biochemicallydefinedautoantibody.
DQB1*02/DRB1*0401/4-DQB1*0302.
DR3/4,(DR3)-DQA1*05-
HLA AND PROGRESSION OF DIABETES-ASSOCIATED AUTOIMMUNITY
3254 DIABETES, VOL. 59, DECEMBER 2010diabetes.diabetesjournals.org

Page 3

cohort comprised older individuals than our subjects, and
the timing of seroconversion could not be defined in the
DPT-1 study.
In the present survey, we were able to start the analysis
from the initial detection of autoantibodies in children
closely monitored from birth onward. After performing the
analysis from the appearance of the second instead of
the first biochemical antibody, the original tendency of the
DR3/DR4 effect disappeared, indicating that the possible
effect might reflect expansion of the autoimmune response
to a more advanced type with more autoantibody speci-
ficities associated with a higher risk.
The strong linkage disequilibrium between alleles in
class I and class II HLA loci emphasizes the importance of
stratification for class II alleles when looking for class I
effects. The major finding in our study—the strong effect of
B*39 on the progression from ?-cell autoimmunity to
clinical disease—was only seen in subjects carrying the
combination of both major class II risk haplotypes, the
DR3/DR4 genotype. We have earlier observed HLA-B*39 to
be common in type 1 diabetes–associated DRB1*0404
haplotypes in Finland (5), and we have also been able to
demonstrate its predisposing effect on type 1 diabetes risk
in DRB1*0404-DQB1*0302–positive subjects in Estonia
and Russia (18). The presence of the HLA-B*39 allele in the
DRB1*08-DQB1*0402 haplotype has also been found to be
a risk factor for type 1 diabetes (3). Extensive single
nucleotide polymorphism analysis has recently confirmed
the independent effect of class I alleles and especially of
the B*39 allele on type 1 diabetes risk (2). In this study,
HLA-B*39 was also, in most cases, found in DRB1*0404-
positive haplotypes, but interestingly the combination
TABLE 2
Cox regression analysis of HLA class I effects on progression to clinical diabetes in children with diabetes-associated autoantibodies
when categorized according to presence of DR3/DR4 class II combination
Follow-up from appearance of first biochemically defined autoantibody*
Multivariate analysis
DR3/4 not present DR3/4 present
HLA-A*03
HLA-A*24
HLA-B*39
0.015
0.096
0.660
0.490 (0.276–0.870)
1.643 (0.915–2.948)
1.208 (0.520–2.805)
0.741
0.142
0.004
1.142 (0.518–2.518)
0.372 (0.099–1.391)
6.564 (1.801–23.926)
Follow-up from appearance of second biochemically defined autoantibody†
Multivariate analysis
DR3/4 not present DR3/4 present
HLA-A*03
HLA-A*24
HLA-B*39
0.003
0.252
0.013
0.388 (0.208–0.724)
1.455 (0.766–2.765)
3.160 (1.279–7.806)
0.554
0.117
0.007
1.310 (0.535–3.206)
0.297 (0.065–1.356)
7.454 (1.740–31.928)
Data are P values and hazard ratio for diabetes progression (95% CI). *Stratified according to age at appearance of first biochemically defined
autoantibody. †Stratified according to age at appearance of second biochemically defined autoantibody.
Time from appearance of 1st AAB (years)Time from appearance of 1st AAB (years)
12 1086420
Probability of T1D Probability of T1D
1.0
0.8
0.6
0.4
0.2
0.0
12 1086420
1.0
0.8
0.6
0.4
0.2
0.0
A
B
B*39 pos.
B*39 neg.
11
53
5
44
1
27
1
21
0
8
0
8
0
1
B*39 pos.
B*39 neg.
17
127
12
100
10
71
6
40
1
17
0
8
0
1
FIG. 1. The effect of the HLA-B*39 allele on the progression to type 1 diabetes after seroconversion to persistent positivity for ICA and at least
one biochemically-characterized autoantibody in children with the HLA-DR3/DR4 combination (A), and children with other class II genotypes (B).
HLA-B*39–positive children indicated by solid line and B*39-negative by dashed line. Kaplan-Meier analysis demonstrated a highly significant
difference between the B*39 positive (n ? 11) and B*39–negative (n ? 53) groups among children carrying the DR3/DR4 combination (P ?
0.00007, log-rank test), but no difference was seen between the HLA-B*39–positive (n ? 17) and –negative (n ? 127) children who did not carry
the high-risk HLA class II combination (P ? 0.768, log-rank test). The panels below the figure show the number of subjects followed at each time
point. AAB, autoantibody; T1D, type 1 diabetes.
K. LIPPONEN AND ASSOCIATES
diabetes.diabetesjournals.orgDIABETES, VOL. 59, DECEMBER 20103255

Page 4

with DR3-positive haplotype was needed because the B*39
effect could not be seen in other genotypes with
DRB1*0404. Among DR3/DR4 heterozygotes, 7 of 11 B*39-
positive samples were DRB1*0404 positive. HLA-B*39
might of course also be associated with the other haplo-
type in these cases, but in a Finnish family trio analysis we
detected B*39 in only 0.9% of 326 (DR3)-DQA1*05-
DQB1*02 haplotypes transmitted to the diabetic child
(unpublished data, J.I.). This difference in B*39 effect
between DR3/DR4 and other genotypes may suggest some
specific interactions between class II and class I.
The genetic constitution of our study population, the
Finnish population in general, and especially the follow-up
cohort derived through HLA DQ-based genetic screening
(7) must also be taken into account when interpreting the
results. Almost all children included in the current analysis
carriedtheDR4-DQB1*0302
DRB1*0401 or DRB1*0404. We are thus unable to distin-
guish between the findings associated with either DR3/
DR4 genotype or DR3 positivity. Because of the genetic
screening criteria applied for the study group, we were
unable to analyze the possible further effects of protective
or neutral class II genotypes on the progression of ?-cell
autoimmunity.
haplotypewith either
ACKNOWLEDGMENTS
This study was supported by the Juvenile Diabetes Re-
search Foundation, the Academy of Finland, the Finnish
Diabetes Research Foundation, the Sigrid Juse ´lius Foun-
dation; by special public grants for medical research at the
Oulu, Tampere, and Turku University Hospitals; and by the
Finnish Cultural Foundation, Varsinais-Suomi Regional
Fund.
No potential conflicts of interest relevant to this article
were reported.
K.L. and Z.G. researched data and wrote the manuscript.
M.K. researched data and contributed to discussion. H.S.
reviewed the manuscript and contributed to discussion.
J.L. researched data and edited the manuscript. R.H., R.V.,
and O.S. reviewed and edited the manuscript and contrib-
uted to discussion. M.K. researched data, reviewed the
manuscript, and contributed to discussion. J.I. researched
data and wrote the manuscript.
Preliminary data from this study were presented in
poster form at the 39th meeting of the Scandinavian
Society for Immunology jointly with the Baltic Immuno-
logical Society, Tallinn, Estonia, 2–5 June 2010.
Parts of this study were presented in poster form at the
11th Congress of the Immunology of Diabetes Society,
Incheon, South Korea, 31 October–3 November 2010.
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