Human Molecular Genetics, 2003, Vol. 12, No.

10 11011110
DOI: 10.1093/hmg/ddg132

Association of a functional 17b-estradiol sensitive

IL6-174G/C promoter polymorphism with
early-onset type 1 diabetes in females
Ole P. Kristiansen1,{, Runa L. Nolse1,{, Lykke Larsen1, Anette M.P. Gjesing1,
Jesper Johannesen1, Zenia M. Larsen1, Anne E. Lykkesfeldt2, Allan E. Karlsen1,
Flemming Pociot1, Thomas Mandrup-Poulsen1,3,*, DIEGG{ and DSGD
1
Steno Diabetes Center, DK-2820 Gentofte, Denmark, 2Department of Tumor Endocrinology, Institute of Cancer
Biology, Danish Cancer Society, DK-2100 Copenhagen , Denmark and 3Department of Molecular Medicine,
Karolinska Institute, SE-17176 Stockholm, Sweden

Received December 16, 2002; Revised and Accepted March 10, 2003

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The type 1 diabetes mellitus (T1DM) candidate gene SNP IL6-174G/C was genotyped in 253 Danish T1DM
families (1129 individuals). TDT analysis demonstrated linkage in the presence of association between the
IL6-174C allele and T1DM in the 416 T1DM offspring, Ptdt 0.04. Gender conditioned TDT analyses revealed
that linkage and association with T1DM were present in females exclusively; Ptdt 6.5 " 10#4 and
Ptdt 2.4 " 10#4, respectively. Random transmission of the IL6-174C/G alleles was found in T1DM males,
non-T1DM males and non-T1DM females; all Ptdt $ 0.37. Heterogeneity analyses (T1DM versus non-T1DM
females) excluded preferential meiotic segregation in females, P 4.6 " 10#3, and demonstrated differences
in the transmission patterns between female and male T1DM offspring, P 5.1 " 10#3. The IL6-174 CC
genotype was associated with younger age at onset of T1DM in females (P 0.002). The impact of 17b-
estradiol (E2) on the IL6-174G/C variants was investigated by reporter studies. The PMA stimulated activity of
the T1DM risk IL6-174C variant exceeded that of the T1DM protective IL6-174G variant by %70% in the absence
of E2 (Pc 0.004), but not with E2 present (Pc 0.12). The PMA stimulated activity of the IL6-174G variant was
repressed without E2 present, but was derepressed by addition of E2, Pc 0.024. In contrast, the PMA
stimulated IL6-174C activity was unaffected by E2 as were the constitutive activities of the IL6-174G/C
variants. In conclusion, higher IL6 promoter activity may confer risk to T1DM in very young females. This
excess risk is negated with increasing age, possibly by the increasing E2 levels in puberty.

INTRODUCTION Interleukin-6 (IL-6) is a pleiotropic cytokine known to be

Human genome-wide scans in type 1 (insulin-dependent)
diabetes mellitus (T1DM) have provided evidence for T1DM as
a polygenic disease (16), but hitherto these scans have not
identified novel etiological T1DM gene variants. T1DM is
considered to be an autoimmune disease (7). Thus, poly-
morphisms in genes encoding proteins with known or inferred
impact on immunity and autoimmunity may be considered
candidate gene variants for T1DM risk.
involved in both the amplification of and protection against the
inflammation in response to infection and tissue injury (8,9).
IL-6 induces expression of hepatic acute phase reactants (9),
stimulates the hypothalamicpituitaryadrenal axis (10), and
induces synthesis of the tissue inhibitor of metalloproteinases-1
(11), the circulating interleukin-1 receptor antagonist and the
soluble tumor necrosis factor p55 (12), actions thought to
mediate mainly anti-inflammatory effects. The IL-6 system
aggravates local inflammation by amplification of leukocyte

*To whom correspondence should be addressed at: Steno Diabetes Center, 2 Niels Steensens Vej, DK-2820 Gentofte, Denmark. Tel: 45 44439101;
Fax: 45 44438232; Email: tmpo@steno.dk
{
The authors wish it to be known that, in their opinion, the first two should be regarded as joint First Authors.
{
The Danish IDDM Epidemiology and Genetics Group.

The Danish Study Group of IDDM in Childhood.

Human Molecular Genetics, Vol. 12, No. 10 # Oxford University Press 2003; all rights reserved
1102 Human Molecular Genetics, 2003, Vol. 12, No. 10
recruitment (13) and in chronic inflammation IL-6 production
contributes to polyclonal B-cell activation and autoantibody
production (14). IL-6 is known to induce Fas expression on
T-cells (15), but also stimulates the expression and delays the
degradation of anti-apoptotic factors (16). Several cell types,
including T-cells, monocytes, fibroblasts, endothelial cells and
pancreatic b-cells synthesize IL-6 (8,17). Hence, inappropriate
regulation of IL-6 may play a role in immune mediated diseases.
The role of IL-6 in the development of T1DM in animal models
is debated. Expression of Il6 under the control of the rat insulin
promoter in transgenic (Tg) mice promotes islet inflammation but
not diabetes in both the diabetes-prone non-obese diabetic (NOD)
mouse (18) and in non-diabetes-prone mouse strains (19).
Continuous IL-6 overexpression in the islets of Langerhans
delays overt diabetes development in the Il6-Tg NOD mouse
(18). Development of autoimmune diabetes or disturbed glucose
homeostasis was not reported when the Il6 gene was universally
expressed in Tg mice (2022). Studies investigating Il6 knockout
NOD mice have not been reported. Hence, local overproduction
of IL-6 promotes islet inflammation, but apparently additional
factors are needed for diabetes development.
The gene encoding IL-6 (IL6, MIM#147620) maps to
chromosome (chr) 7p21 in man (23) and chr 5 in the mouse
(24). The murine Il6 maps to Idd15, a region that confers
susceptibility to diabetes in the NOD mouse (25). None of the
human genome-wide scans (16) have identified this region as
a T1DM susceptibility region in unconditioned analyses. In the
Scandinavian T1DM families a peak-LOD of almost one was
found in the region harbouring IL6 (5). Furthermore, gender
stratified analysis (26) of 356 T1DM UK sib-pairs (3) identified
differences in LOD scores between the markers of chr 7.
However, this difference was not significant for the marker
D7S629 mapping %47 kb downstream of the 30 region of IL6.
Recently, a casecontrol study in UK type 1 diabetics demons-
trated association between T1DM and the IL6-174G/C
promoter polymorphism (27). Interestingly, this polymorphism
has been suggested to be of importance for IL6 promoter
activity based on in vivo serum IL-6 levels (2830) and reporter
assay studies (28), although these findings have been
challenged (31,32). The polymorphism has been shown to be
associated to other autoimmune diseases (28,3335). Finally,
the activity of the IL6 promoter has been shown to be
suppressed by 17b-estradiol (E2) (36,37). However, none of the
studies have investigated the effect of E2 on IL6 promoters
harbouring different allelic variants of the IL6-174G/C
promoter polymorphism.
Hence, in order to evaluate the role of this polymorphism in
T1DM we genotyped a T1DM cohort comprising 253 Danish
Caucasian nuclear T1DM families (1129 individuals) and by
transmission disequilibrium testing (TDT) found evidence for
linkage and association of the IL6-174G/C variation and T1DM,
but exclusively in females. Furthermore, the IL6-174 CC
genotype was associated with younger age at onset in T1DM
females. Consequently, we evaluated the influence of E2
preincubation on the constitutive and PMA treated promoter
activity of the two IL6-174G/C promoter variants in reporter
assays. We show that the PMA stimulated activity of the
T1DM predisposing IL6-174C variant was %70% higher than
the protective IL6-174G promoter variant in the absence of E2,
but the inability of PMA to induce IL6-174G promoter activity
in the absence of E2 was restored by preincubation with E2,
suggesting that the IL6-174C variant associated risk in young
females is conferred by high IL6 promoter activity, an effect
that is negated by increasing E2 levels in puberty.
RESULTS
Genotyping data and TDT analysis of the IL6-174G/C
SNP in affected and unaffected offspring
In the parents we found a heterozygosity index of 49.6% (230
of 463 parents) and IL6-174G and IL6-174C allele frequencies
of 54.4 (504/926) and 43.6% (422/926), respectively. The
variation was found to be in HardyWeinberg equilibrium.
The TDT analysis of transmission to the 416 T1DM offspring
revealed a significantly increased transmission of the IL6-174C
allele in affected offspring; Ptdt 0.04, Table 1. Importantly,
random transmission of the two alleles was found in the
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unaffected offspring, Table 1.
Evaluation of the effect of HLA-risk (and putative interaction
with IDDM1) was assessed by stratification of affected offspring
in groups of high HLA-risk (HLA-DR3/4) and non-high
HLA-risk (HLA-non-DR3/4). TDT analysis in the two HLA
subsets of affected offspring did not reach statistical significance
and no difference in the transmission pattern between groups
was observed (Table 1). Hence, an interaction between IDDM1
and the IL6-174G/C SNP was not observed.
TDT analysis in female and male offspring
Interestingly, stratification of the offspring by gender revealed
profound differences in the transmission patterns between male
and female T1DM offspring (Table 2). In female T1DM
patients a highly significant increased transmission of the
IL6-174C allele was found, Ptdt,c 0.004 (corrected for n 6
comparisons; Table 2). Importantly, random transmission of the
IL6-174G/C variants was found in non-T1DM female off-
spring, excluding a putative transmission distortion ratio for
this variation in all female offspring (Table 2). The transmis-
sions in T1DM and non-T1DM female offspring also displayed
differences by heterogeneity analysis; P 0.0046, Pc 0.018
(corrected for n 4 comparisons), substantiating the finding in
T1DM females. All the above findings indicate that the
IL6-174G/C polymorphism is linked in the presence of
association to female T1DM in the cohort investigated. By
identification of the female index-cases (defined as the first
female offspring to have T1DM in each family, and thus only
one female offspring in each family included, n 165)
association of the IL6-174G/C polymorphism with T1DM in
females was demonstrated, Ptdt,c 1 " 10#3 (Table 2). This
pattern was not found in the 168 male index cases (defined as
above for the T1DM female offspring; Table 2).
The surplus of 37 IL6-174C transmissions found in the initial
TDT analysis in all T1DM offspring (Table 1) all originated
from the T1DM female offspring group in which a surplus of
44 IL6-174C transmissions was observed (Table 2). In male
T1DM and non-T1DM offspring random transmission was
observed (Table 2). Heterogeneity analysis of the transmission
patterns in T1DM males and females also demonstrated
 Human Molecular Genetics, 2003, Vol. 12, No. 10 1103

Table 1. Transmission of IL6-174G/C alleles in Danish T1DM families

Group Number in group Transmitted allele w2,tdt (1 d.f.) Ptdt

IL6-174G IL6-174C [Mean; 95% CI]
All affected 416 143 180 [0.56; 0.510.61] 4.24 0.04
All unaffected 250 104 101 [0.49; 0.420.56] 0.04 0.84
Affected HLA DR3/4 157 59 78 [0.57; 0.490.65] 2.63 0.10
Affected non-HLA DR3/4 246 77 95 [0.55; 0.480.62] 1.88 0.17

Comparison of the transmission pattern between (1) all affected and unaffected, P 0.15, and (2) HLA groups, P 0.76.

Table 2. Transmission of IL6-174G/C alleles in female and male offspring

Gender T1DM status Number in group Transmitted allele w2,tdt(1 d.f.) Ptdt
IL6-174G IL6-174C [Mean; 95% CI]
Female T1DM 200 61 105 [0.63; 0.560.70] 11.63 0.00065
Unaffected 127 56 47 [0.46; 0.360.55] 0.79 NS
T1DM index cases 165 47 90 [0.66; 0.580.74] 13.50 0.00024
Male T1DM 216 82 75 [0.48; 0.400.56] 0.312 NS

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Unaffected 123 48 54 [0.53; 0.430.63] 0.35 NS
T1DM index cases 168 64 61 [0.49; 0.400.58] 0.07 NS

Comparison of the transmission pattern in (1) affected and unaffected female offspring, P 0.0046, and (2) affected female and affected male offspring, P 0.0051.

differences between the two groups (all cases, P 5.1 " 10#3,
Pc 0.02; and index-cases, P 5.7 " 10#3, Pc 0.023),
substantiating the finding that linkage and association of the
IL6-174G/C polymorphism with T1DM were phenomena
exclusively related to the female offspring.
We identified families with female offspring in which one
parent was homozygous (CC or GG) and the other parent was
heterozygous. This approach allowed us to evaluate both the
parental transmission and the putative effect of the non-
informative (homozygous) parents genotype, albeit signifi-
cantly reducing the number of informative meioses. In families
with one CC-homozygous and one CG-heterozygous parent the
distortion of the informative parents transmissions to both
T1DM (n 33) and non-T1DM (n 16) female offspring was
profound; 26 of the 33 informative transmissions to T1DM
female offspring were IL6-174C alleles (79%; 95% CI 6593%,
Ptdt 9 " 10#4, Ptdt,c 3.6 " 10#3, corrected for n 4 compar-
isons), whereas only three of 16 informative transmissions to
non-T1DM female offspring were IL6-174C alleles (19%; 95%
CI 038%, Ptdt 0.0124, Ptdt,c 0.05). Heterogeneity analysis
of the transmission pattern between the T1DM and non-T1DM
female offspring in these families revealed highly significant
differences; P 2.2 " 10#4, Pc 8.8 " 10#4 (corrected for
n 4 comparisons). In families with one GG homozygous
parent and one GC heterozygous parent the transmission
pattern was not distorted; 29 of 49 (Ptdt 0.2) and 16 of 33
transmissions to T1DM and non-T1DM females, respectively,
were IL6-174C transmissions. Despite the low number infor-
mative transmissions in the two groups (GG/GC versus CC/GC
parents) a borderline significant (uncorrected) difference in
transmission pattern to T1DM females was observed, P 0.064.
Hence, an effect of both the genotype of the TDT non-
informative parent (IL6-174G protects) and a profound effect of
the transmitted allele from the TDT informative parent were
observed; IL6-174C and IL6-174G confer risk and protection,
respectively, if the other parent is CC homozygous. Such effects
were not observed in male offspring (data not shown).
The IL6-174 CC genotype associates with young age at
onset in female T1DM offspring
The genotypes of the IL6-174G/C SNP have been shown to
affect the age of onset in other diseases (33,38). Thus, we
evaluated the effect of the IL6-174G/C variant on the age at
onset of T1DM by evaluating both the transmission pattern to
T1DM offspring above and below the median age at onset in
the cohort, and by evaluating the effect of gender, genotype and
interaction terms of the two (categorical, linear and recessive
effect of the C-allele) on the age at onset in index T1DM male
and female offspring by two-way ANOVA.
In the young onset T1DM offspring (<11.1 years, n 208)
increased IL6-174C transmission was observed since 58% (107
of 186 informative transmissions; 95% CI 5165%; Ptdt 0.04)
were IL6-174C transmissions. Random transmission was
observed in the high age at onset group: 64 IL6-174G and 73
IL6-174C transmissions, respectively. However, no significant
difference in transmission patterns between the two age-at-onset
groups was found; P 0.45.
In order to minimize a familial effect on the age at onset only
the 165 index female and the 168 index male offspring were
included. The mean and median ages of onset in T1DM
female and male offspring grouped by IL6-174G/C genotype are
illustrated in Figure 1. The overall median age of onset in the
male and female index cases demonstrated significantly younger
onset of T1DM in the female group; male versus female median;
12.20 versus 9.30 years, P 2.8 " 10#3. The fitted two-way
ANOVA demonstrated an effect of gender on age at onset;
P 1.5 " 10#3. This analysis did not show an overall effect of
the genotype on the age at onset; P 0.43. The categorical and
linear interaction between gender and genotype on the age at
1104 Human Molecular Genetics, 2003, Vol. 12, No. 10
Figure 1. Mean and median age at onset in IL6-174G/C genotype stratified
female end male T1DM index cases. Age at onset of T1DM (means and
95% CI) for each sex and genotype as estimated by the ANOVA interaction
model. The horizontal lines indicate quartiles of onset ages. Numbers in each
group: femalesCC (n 47), CG (n 76) and GG (n 42), respectively;
malesCC (n 39), CG (n 77) and GG (n 52), respectively.
onset was not significant; F(2,327) 1.56, P 0.21 and
F(1,328) 1.64, P 0.20, respectively. In the recessive-based
model a trend for interaction of gender and genotype was
observed [F(1,328) 3.07, P 0.08], suggesting that the
C-allele in a recessive way may affect age at onset differently
in males and females. In the interaction model the age at onset
was significantly different between males and females with the
CC-genotype, (means: (males), 14.04, 95% CI 11.6216.46 and
females, 8.81, 95% CI 6.6011.01; P 0.002; Fig. 1), thus,
suggesting a recessive effect of the C-allele on age at onset in
females but not in boys (Fig. 1). The effect of the genotype and
gender on age at onset was clearly demonstrated by the
cumulative distribution function of age in the six genotype/
gender groups (Fig. 2). This figure clearly visualizes the
differences in age at onset of T1DM between the IL6-174 CC
males and females. Taken together, the above analyses suggest
that age at onset of T1DM in females is affected by the IL6-174
CC genotype. Importantly, the observations also suggest that the
difference in age at onset between boys and girls is partly
explained by the fact that girls carrying the CC genotype had
lower age of onset.
The IL6-174C promoter variant determines higher
stimulated but not constitutive promoter activity in the
Ishikawa cell line in the presence of 17b-estradiol
The human Ishikawa endometrial adenocarcinoma cell line was
demonstrated to express the human estrogen receptor (hER) by
western blotting (data not shown). We observed significant
differences in activity between the four conditions (constitutive
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Figure 2. Cumulative distribution function of age at onset of T1DM in
males and females stratified for IL6-174G/C genotype. Numbers in each
group: femalesCC (n 47), CG (n 76) and GG (n 42), respectively;
malesCC (n 39), CG (n 77) and GG (n 52), respectively.
and PMA-treated activities of the two different construct
variants without E2 preincubation; P 1.7 " 10#3; Fig. 3). The
PMA stimulated activity of the 225IL6C-pGL3 construct
exceeded the PMA stimulated activity of the 225IL6G-pGL3
construct; Pc 0.038 (Fig. 3). PMA treatment did not stimulate
the promoter activity of the 225IL6G-pGL3 construct. The
constitutive activities of the two construct variants displayed no
significant differences.
PMA stimulates the IL6-174G promoter in the presence of
17b-estradiol
A significant difference in activity between the four conditions
with E2 preincubation was demonstrated (P 0.048; Fig. 3).
The PMA stimulated activities of the two construct variants
displayed no significant difference; Pc 0.12.
The PMA-treated activity of the two construct variants with
and without E2 preincubation was evaluated and significant
differences were demonstrated; P < 6.7 " 10#5 (Fig. 3). The
main cause of this highly significant difference was the low
activity of the 225IL6G-pGL3 construct in the absence of
E2 preincubation (Fig. 3). This difference was corrected by the
presence of E2, Pc 0.024 (Fig. 3). No differences in
constitutive activities between of the two construct variants with
and without E2 preincubation were observed, P 0.33 (Fig. 3).
Evaluation of the PMA stimulation indices (calculated as the
ratio of mean PMA stimulated activity :mean constitutive
activity of the construct in each experiment) of the two-
construct variant with and without E2 preincubation (Fig. 4) by

Figure 3. Constitutive and PMA stimulated activity of 225IL6G/C-pGL3 con-
structs transfected into Ishikawa cells grown with and without 17b-estradiol.
Black (225IL6G-pGL3) and white (225IL6C-pGL3) bars are mean promoter
activity (estimated as described in Methods, n 5). Error bars are 95% CI.
P-values (paired-t-test for means) are corrected for multiple testing.
ANOVA revealed significant differences between the condi-
tions; P 0.04. The stimulation indices of the 225IL6G-pGL3
(E2), the 225IL6C-pGL3 (E2) and the 225IL6C-pGL3
(#E2) constructs were identical and all exceeded the stimula-
tion index of the 225IL6G-pGL3 construct stimulation index
without of E2 present (Fig. 4).
In summary, PMA treated but not constitutive promoter
activity was %70% higher for the IL6-174C allele variant
compared with the IL6-174G variant in the absence of E2.
Interestingly, the inability of PMA to stimulate the IL6-174G
promoter variant and the stimulatory capacity of the IL6-174G
allele was corrected by addition of E2.
The NOD and eight non-diabetic mouse strains all carry
the Il6 G promoter variant
Sequence analysis comparison of the #275 to 50 region
(M20572: nt 10031350) of Il6 in the NOD (NOD/Lt) mouse
strain and eight non-diabetic mouse strains did not reveal any
difference between the strains. The sequence in all strains
investigated aligned 100% to the published sequence
(M20572), equal to the G allele variant.
DISCUSSION
We demonstrate evidence for significant linkage in the presence
of association between the IL6-174G/C SNP and T1DM in a
Human Molecular Genetics, 2003, Vol. 12, No. 10 1105
Figure 4. Stimulation indices of 225IL6G/C-pGL3 constructs transfected into
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Ishikawa cells grown with and without 17b-estradiol. Black (225IL6G-pGL3)
and white (225IL6C-pGL3) bars are mean stimulation indices of the constructs
(estimated as described in the text, n 5). Error bars are 95% CI.
Danish cohort of 253 T1DM families by use of TDT analysis.
Highly significant linkage (TDT analysis in all female T1DM
offspring) and association (TDT analysis in index females only)
were present in females exclusively, with 63 and 66% IL6-
174C transmissions, respectively. Random transmission was
observed in males. Preferential meiotic segregation as a cause
of the above finding in females was ruled out, as random
transmission was found in unaffected female offspring, and in
addition significant heterogeneity in the transmission patterns
between affected and unaffected female offspring was demon-
strated. In contrast to the observation of a previous case-control
study of association between T1DM in both sexes and the IL6-
174 GG-genotype and the IL6-174G allele (27), we found
preferential transmission of the IL6-174C allele in the Danish
T1DM females. However, exploratory gender stratified sib-pair
analysis (26) in the UK dataset (3,26) was not able to
demonstrate linkage of the markers D7S493 and D7S629 to
T1DM in 82 femalefemale sib-pairs, although the latter
marker was nominally more linked in female than males. The
UK study (27) also evaluated IL6-174G/C transmission in 53
T1DM trio families (gender of T1DM offspring not reported),
and did not find distorted transmission of the IL6-174G and
C alleles in these families with an IL6-174 C allele 95% CI
for transmission of 3258%. This is not different from the
IL6-174C transmission (95% CI 5161%) observed in our
material when analysing transmission to all T1DM offspring
(Table 1). An estimate of the transmission distortion to the
cases in the UK study (27), using a genotype distribution in the
parents of the T1DM cases similar to that in the controls,
suggests a transmission pattern with 7989% IL6-174G allele
transmissions. This is significantly different from that observed
in the T1DM trios (27). Further, the UK study did not report
gender-stratified analyses (27). A very likely caveat of the latter
study (27) is that of spurious association due to population
admixture and selection bias (39); the main issues of concern
are that the control subjects were sequential newborn babies,
1106 Human Molecular Genetics, 2003, Vol. 12, No. 10
born approximately a mean of 30 years later than the T1DM
cases (population admixture?), born by normal delivery
(selection?) and born by mothers having no familial history
of diabetes (selection?). Finally, the numbers of individuals in
the casecontrol study groups were small. It is most unlikely
that the association of T1DM with different alleles in the
present and the UK casecontrol study (27) reflects oppositely
directed linkage disequilibrium (LD) with a closely linked
culprit T1DM risk gene variant. TDT analysis of candidate
gene variations with minor impact is more powerful than sib-
pair-based analyses (40), and the TDT analysis does not suffer
from risk of spurious association as do genetic casecontrol
studies using small samples (39). Further, we demonstrated
highly significant intra-familial heterogeneities in the transmis-
sion patterns between affected males and females and between
affected and unaffected female offspring.
Our observation outlines the necessity of evaluating genetic
linkage and association data in T1DM not only in analyses
grouped by HLA class II genotype or HLA class II sharing
(41,42), but also stratified by gender in order to identify genes
that confer T1DM risk, in particular when analysing genes and
gene variations or markers close to genes that are likely to be
affected by the gender of the affected.
As expected we found the age at onset to be lower in the
female offspring and demonstrate that the reason for this male-
female difference in age at onset mayat least in partbe
explained by the significantly lower age at onset in the IL6-174
CC homozygote females compared with CC males. An effect
of the IL6-174G/C genotype on age at onset was also observed
in patients with rheumatoid arthritis (33).
Other variants are found in IL6 promoter and 30 UTR (32,43).
These include a frequent #597A/G SNP, a rare #572G/C SNP,
a #373AnTm (nomenclature as in 32) and a highly polymorphic
VNTR in the 30 -UTR, with three frequent repeat variants
denoted 3, 4 and 7 (43). Interestingly, in a Scottish population
(that have the same genotype distribution as observed in the
parents in our study), the IL6-174C allele was found in the
promoter haplotype 597A-572G-373A8/T12-174C in 63 of
65 haplotypes (43) in accordance with the observation of this
haplotype in 28 of 29 UK individuals (32). This haplotype
(denoted IL6.0103) is in LD with the VNTR variant 3 and was
found in 60 of 65 haplotypes including the IL6-174 C allele
(43). The IL6-174G allele combines in %92% of all haplotypes
with the #597G and #572G alleles (32,43). This haplotype
does not harbour the #373A8/T12 variant (32) but several other
#373AnTm variants, and is found mainly with of the VNTR
variants 4 (denoted IL6.0204) and 7 (IL6.0207) (43). Thus, by
genotyping for IL6-174G/C SNP and demonstrating associa-
tion with the IL6-174C allele we have most likely demonstrated
association between T1DM in females and the IL6.0103
haplotype and the genetic variants in this haplotype, apart
from the #572G/C SNP. On the other hand, we cannot exclude
these variants or the IL6.0103 haplotype per se as the
etiological T1DM gene variant(s). However, only the IL6-
174G/C variants were included in the constructs demonstrating
significant functional impact of E2.
In hER-positive cells, E2 has been claimed to negatively
affect stimulated activity of the IL6 promoter (36,37,44).
Reporter constructs holding the different variants of the
IL6-174G/C SNP were found to display differences in LPS
and IL-1b stimulated promoter activity with higher stimulated
activity of the IL6-174G construct compared to IL6-174C
construct (28). This observation was challenged by the finding
of marginally increased activity of the IL6-174C constructs
compared with IL6-174G constructs in truncated (#211 to
13) IL6 promoter constructs (32). In light of the above studies
and our TDT data we were prompted to investigate the
hypothesis that the activity of the two IL6-174G/C SNP
variants was differently affected by E2. We found that the
inability of PMA to stimulate the IL6-174G variant was
corrected by E2 preincubation whereas the PMA stimulated
activity IL6-174C promoter variant was unaffected by E2. We
did not confirm the previously reported decreased IL6 promoter
activity by E2 in Ishikawa cells (37), which may be due to hER
down regulation (37), or differences in assay conditions. The
SNP map to a negative regulatory domain (#224 to #158 bp)
in the IL6 promoter (45). This site, however, has not been
directly implicated in the E2 regulation of IL6 promoter activity,
which is most likely mediated through a direct binding of
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NF-IL6 and NF-kB to the hER, thereby preventing these
transcription factors from binding to the more downstream
IL6 promoter (36,37). In vitro exposure of lymphocytes and
monocytes to PMA stimulates and inhibits IL6 expression,
respectively (46). Hence, in vivo regulation of IL6 promoter
activity in various immune competent cells is likely to be
cell-dependent.
The great homology (>80% in the 300 bp upstream of the
transcription start sites) of the 50 flanking regions of the human
and the murine IL-6 genes (47), the present observation of
association of the IL6-174C variants in female T1DM patients
only, the mapping of Il6 in the Idd15 region in the NOD mouse
(25) and increased incidences of diabetes in castrated male NOD
mice (48) prompted us to sequence and compare the Il6 #275 to
50 region in NOD (NOD/Lt) mice with eight non-diabetic
mouse strains. We found complete sequence homology between
all strains, and hence exclude a genetic variant in this part of the
promoter as the Idd15 etiological variant in the NOD mouse and
that a SNP homologous to IL6-174G/C exists in mice.
In conclusion, we have demonstrated that the IL6-174C
promoter variant is highly associated with T1DM in Danish
females, but not in males, and that the association is not caused
by preferential transmission distortion in females. This is the
first observation of such a gender difference in family based
studies of association between a gene variation proper and
T1DM. The observation underscores the need for analysis of
linkage and association in gender stratified groups in T1DM.
Furthermore, our study highlights the importance of avail-
ability of informative unaffected offspring to exclude meiotic
segregation distortion. We also demonstrate for the first time by
reporter assay studies evidence suggesting that the repressed
PMA stimulated activity of the IL6-174G variant is reverted by
E2, whereas the stimulated activity of the IL6-174C variant is
E2-insensitive and higher than the stimulated activity of the
IL6-174G variant in the absence of E2 present. Although, our
study does not provide a direct mechanistical explanation for
the role of the IL6-174G/C SNP variants in T1DM develop-
ment, it may suggest that higher IL6 promoter activity may
confer risk to T1DM in very young females. This excess risk is
negated with increasing age, possibly by the increasing E2
levels in puberty.

MATERIAL
Two-hundred and fifty-three Danish Caucasian nuclear type 1
diabetes families with a total of 1129 individuals were
genotyped. The material comprised: (1) 150 sib-pair families
in which both parents in 107 families and one parent in 43
families were available for genotyping; and (2) 103 simplex
families. The index cases defined as the first affected offspring in
each family (128 females), had an onset of T1DM prior to age
30 years (mean age at onset ' SD 10.1 ' 6.7 years, median age
at onset 9.2 years). A total of 416 affected (200 females) and
250 unaffected (126 females) offspring were typed. Mean and
median ages at onset for all affected were 12.9 and 11.1 years
(range 045), respectively. All families were unrelated.
METHODS
Genotyping for the IL6-174G/C polymorphism
This was performed by a PCR based RFLP assay. 164 bp
(position 9391102, GenBank accession no. Y00081.1; #222
to #59 relative to the transcription start site) (47) of the IL6
promoter were amplified using the primers 50 -GCC TCA ATG
ACG ACC TAA GC-30 and 50 -TCA TGG GAA AAT CCC
ACA TT-30 . The PCR mixture comprised 50 mM of each dNTP
(Life Technologies, Paisley, UK), 1.5 mM MgCl2, 0.5 U Taq-
polymerase (Life Technologies), 1 mM of each primer and
1 " polymerase buffer (Life Technologies) and 40 ng genomic
DNA in total reaction volume of 20 ml. Cycling conditions
were: one cycle of 5 min at 95( C followed by 37 cycles of 30 s
at 95( C, 30 s at 58( C and 30 s at 72( C and finally one cycle of
10 min at 72( C. The product was digested by 2 U of NlaIII
(New England BioLabs, Beverly, MA, USA) for 16 h at 37( C.
The amplified G allele variant was digested once corresponding
to position #59 bp leaving one large fragment of 163 bp,
whereas the C allele was cleaved twice corresponding to the
positions #170 and #59 resulting in two fragments of 111 and
52 bp, respectively. The digested fragments were applied to 2%
LE-agarose gels containing 0.5 mg ethidiumbromide (Sigma,
St Louis, MO, USA) per ml gel. The assay performance was
evaluated by blinded randomised re-genotyping of 80 samples
and complete agreement was found.
Sequencing of the mouse IL6 promoter
The #275 to 50 region (M20572: nt 10031350 ) of IL6 in
NOD (NOD/Lt) mice and eight non-diabetic mouse strains
(C57BL/6J, NOR/Lt, SWR/Bm, SJL/Bm, ALS/LtJ, ALR/
LtJ, FVB/NJ, NON/Lt) was directly sequenced using the
primers 50 -TTC CCA TCA AGA CAT GCT CA-30 (forward)
and 50 -GCA AGG AAC TGC CTT CAC TTA-30 . Hence,
amplicons spanning the mouse Il6 gene from position #302 to
90 relative to the transcription start site were produced for
sequencing. All mouse DNAs were obtained from The Jackson
Laboratory (Bar Harbor, ME, USA).
Cloning of the Luciferase-reporter constructs
The two allelic forms of the IL6-174G/C SNP were cloned into
the pGL3 Basic Vector (Promega, Madison, WI, USA) by
Human Molecular Genetics, 2003, Vol. 12, No. 10 1107
introduction of IL6 promoter insert variants ranging from
positions #225 to 24 bp relative to the transcription start site
(47). The constructs were denoted 225IL6C-pGL3 and
225IL6G-pGL3. The cloning procedure was performed by
initial PCR amplification of genomic DNA from subjects
homozygous (CC and GG) for the IL6-174 variants using the
primers 50 -CCT GCA AGA GAC ACC ATC CT-30 (forward)
and 50 -TCC TGG AGG GGA GAT AGA GC-30 (reverse)
amplifying the #1153 to 54 region GenBank accession no.
Y00081; 81214) of the IL6 promoter. This was followed by
nested PCR using the primers 50 -GGG GTA CCC CGA CAC
CAT CCT GAG GGA AGA (forward) and 50 -GGG AAG CTT
CCC GGT GGC TCG AG (reverse) and introducing KpnI
and HindIII restriction sites (attached nucleotides in bold and
restriction sites in italics) in the 50 and 30 ends, respectively, in
an amplicon comprising the #1144 to 24 bp region of the
promoter (GenBank accession no. Y00081; 171184).
Restriction cleavage of the amplicons and the pGL-3 Basic
Vector using the KpnI and HindIII (both from New England
Downloaded from http://hmg.oxfordjournals.org/ by guest on June 3, 2015
Biolabs) restriction enzymes was performed. The cleaved pro-
ducts were ligated directly into the similarly cleaved and
dephosphorylated pGL3 Basic Vector using the Original TA
cloning1 kit (Invitrogen, Carlsbad, CA, USA). Promoter
positive clones were identified by use of the primers 50 -GCC
AGA ACA TTT CTC TAT CG-30 (forward, denoted 5200) and
50 -TAC CAA CAG TAC CGG AAT GC-30 (reverse, denoted
5201). Promoter positive constructs were transfected into One-
Shot1 cells as detailed by the manufacturer (Invitrogen).
Plasmid DNA (pDNA) amplification was performed using the
Qiagen Mega-2500 kit (Qiagen GmbH, Hilden, Germany).
Plasmid quality was assessed by DNA purity evaluation by
spectrophotometry at 280 and 260 nm (a ratio >1.9 was
considered acceptable), and by gel-evaluation for nicked and
denatured pDNA. Four constructs (two holding the IL6-174C
and two holding the IL6-174G variants) were selected for
further processing. The four constructs were cleaved by the
restriction enzymes Acc65I (Promega) and NheI (New England
Biolabs) using the manufacturers instructions, cleaving the
product at positions #1141 and #225 in the inserted IL6
promoter inserts. Constructs were isolated using 0.5%
Ultrapure gels and subsequent purification by centrifugation
in Ultra-Free DA tubes (Millipore no. 42600). The ends of the
isolated constructs were blunted using the Klenow reagent and
re-ligated using the T4-DNA-ligase (Invitrogen) following the
manufacturers instructions. Control PCR of the re-ligated
225IL6G/C-pGL3 constructs was performed using the primers
5200 and 5201. Plasmid DNA and control of plasmid DNA
was performed as described above and DNA concentration was
assessed by spectrophotometry in at least two different
dilutions of each construct. Finally, the IL6 promoter inserts
of the four selected high quality constructs were sequenced
using the primers 5200 and 5201.
Western blotting
Expression control of the human estrogen receptor (hER) in the
human endometrial adenocarcinoma Ishikawa cell line (in which
PMA has been shown to induce IL6 promoter activity and E2 to
reduce PMA stimulated IL6 promoter activity by 2040% without
1108 Human Molecular Genetics, 2003, Vol. 12, No. 10
need to transfection of the hER vector) (37) was performed by
western blotting analysis as described previously (49).
Cell culture
The Ishikawa cell line (a generous gift from B. Sehested-
Hansen, Novo Nordisk A/S) was cultured in DMEM (Gibco,
Paisley, Scotland) supplemented with non-essential AA
(Gibco), sodium-bicarbonate (Gibco) 2.2 g/l final concentra-
tion, L-glutamine (Gibco) 2 mM final concentration, penicillin
and streptomycin (Gibco) and 10% FCS (Gibco), (named
CM1). The cell line was grown at 37( C in 5% CO2 at an initial
cell concentration of 2 " 106 cells/30 ml CM1 in T175 culture
flasks. The culture medium was renewed bi-daily and cells were
split twice weekly by standard methods. Three days prior to
transfection the cells were split, washed and transferred to a
steroid-free (double-charcoal stripped FCS and phenol-red free)
media (CM2) consisting of DMEM without phenol red (Gibco)
with 2.2 g/l Sodium bicarbonate (Gibco), 4 mM L-Glutamine
(Gibco), penicillin and streptomycin (Gibco), 3.5 g/l -D-
glucose (total concentration in medium 4.5 g/l, Sigma) and
5% double-charcoal stripped FCS (Gibco) at an initial cell
concentration 2 " 106 cells/30 ml CM2 in T175 culture flasks.
Twenty hours prior to transfection the cells were split, washed
and transferred to 24-well trays (25 000 cells in 500 ml CM2/
well). On the day of transfection the medium was removed and
replaced by 400 ml CM2.
Transfection and stimulation procedure
One microgram of plasmid DNA 95% IL6-225(G or C)pGL3-
vector construct and 5% Tyrosine Kinase-Promoter Renilla
Luciferase internal-vector control (TK-pRL, Promega) and 2 ml
SuperfectTM (Qiagen) in a total volume of 100 ml
were incubated for 15 min at room temperature according to
the manufacturers instruction and then added to each well. The
Ishikawa cells were incubated for 4 h in the presence of the
plasmid DNA and the SuperfectTM and the medium was
aspirated. All constructs were set up in triplicate for each of the
conditions listed below, and two different preparations of each
construct variant were included in all experiments (n 5) to
minimize the putative effect of the variation between individual
plasmid preparations. The medium was replaced by
CM2 10 nM E2 (Sigma no. 2758) or CM2 vehicle
(ethanol) and the cells were incubated for 20 h. The medium
was then removed and replaced by CM2 with or without
100 ng/ml PMA for 24 h. Hence, for each construct four
conditions were analysed: (1) #E2/#PMA; (2) #E2/PMA;
(3) E2/#PMA; (4) E2/PMA. Thus, the constitutive and
PMA stimulated activity of the two different constructs were
evaluated with and without preincubation with E2.
Luciferase reporter assay
The Ishikawa cells were washed once in one ml PBS, 125 ml
passive lysis buffer (Promega) was added and the cells were
incubated at RT for 30 min shaking at 500 rpm. Quantitation
of luminescence was performed using the Dual-LuciferaseTM
kit (Promega) and a Lumat LB 9507 luminometer (EG&G
Berthold), following the manufacturers instructions.
In order to reduce the inter-assay variation resulting from the
luciferase reagents, the activity of the 225IL6C-pGL3 and
225IL6G-pGL3 variants (corrected for transfection efficacy by
the co-transfected TK-pRL) was expressed as fold activity
compared with activity of the pGL3 Basic vector.
Statistical analysis
Transmission disequilibrium testing was performed using the
Sib-TDT analysis software (50). The 95% confidence interval for
transmission distortion was calculated using the method
described in (51). Heterogeneity analysis of transmission patterns
between groups (w2-analyses, Yates corrected when appropriate)
and MannWhitney analysis of median age at onset in male and
females were performed using the MEDSTAT software Version
2.1 (Astra, DK). For the age-at-onset data we fitted a two-way
ANOVA with gender and genotype, and three different interaction
terms between gender and gene-dose (categorical, linear and
recessive effect of the IL6-174C allele) using the web-based
Downloaded from http://hmg.oxfordjournals.org/ by guest on June 3, 2015
statistical software R (www.r-project.org). Statistical analyses of
the reporter assay data were performed by initial F-testing for
variance compatibility between groups followed two-way
ANOVA between relevant groups and subsequent paired t-tests
(Excel, Microsoft, USA). Five percent was considered the limit of
significance. Correction for multiple testing was performed by
the Bonferroni method.
ACKNOWLEDGEMENTS
We appreciate the work of The Danish Study Group of Diabetes
in Childhood (DSGD) and the Danish IDDM Epidemiology and
Genetics Group (DIEGG) in family collection. For participating
departments in DSGD and DIEGG see Larsen et al. (52). We are
grateful for the biostatistical support from Bendix Carstensen
and the technical assistance of Anette Hellgren Adamsen, Anna
Hlin Schram and Marja Deckert. The antibody against K7 was a
gift from Jiri Bartek, Danish Cancer Society. The Danish
Diabetes Association, The Poul and Erna Sehsted Hansen
Foundation and Novo Nordisk A/S supported this work. O.P.K.
was a recipient of Research Fellowship from JDRFI (JDRFI
grant no. 3-1999-21). The Danish National Ethics Committee
approved the study.
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