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DOI: 10.1093/hmg/ddg132
Received December 16, 2002; Revised and Accepted March 10, 2003
*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.
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The Danish IDDM Epidemiology and Genetics Group.
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 in the absence of E2 was restored by preincubation with E2,
contributes to polyclonal B-cell activation and autoantibody suggesting that the IL6-174C variant associated risk in young
production (14). IL-6 is known to induce Fas expression on females is conferred by high IL6 promoter activity, an effect
T-cells (15), but also stimulates the expression and delays the that is negated by increasing E2 levels in puberty.
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 RESULTS
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 Genotyping data and TDT analysis of the IL6-174G/C
is debated. Expression of Il6 under the control of the rat insulin SNP in affected and unaffected offspring
promoter in transgenic (Tg) mice promotes islet inflammation but
not diabetes in both the diabetes-prone non-obese diabetic (NOD) In the parents we found a heterozygosity index of 49.6% (230
mouse (18) and in non-diabetes-prone mouse strains (19). of 463 parents) and IL6-174G and IL6-174C allele frequencies
Continuous IL-6 overexpression in the islets of Langerhans of 54.4 (504/926) and 43.6% (422/926), respectively. The
delays overt diabetes development in the Il6-Tg NOD mouse variation was found to be in HardyWeinberg equilibrium.
(18). Development of autoimmune diabetes or disturbed glucose The TDT analysis of transmission to the 416 T1DM offspring
homeostasis was not reported when the Il6 gene was universally revealed a significantly increased transmission of the IL6-174C
expressed in Tg mice (2022). Studies investigating Il6 knockout allele in affected offspring; Ptdt 0.04, Table 1. Importantly,
NOD mice have not been reported. Hence, local overproduction random transmission of the two alleles was found in the
Comparison of the transmission pattern between (1) all affected and unaffected, P 0.15, and (2) HLA groups, P 0.76.
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
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, respectively, if the other parent is CC homozygous. Such effects
Pc 0.02; and index-cases, P 5.7 " 10#3, Pc 0.023), were not observed in male offspring (data not shown).
substantiating the finding that linkage and association of the
IL6-174G/C polymorphism with T1DM were phenomena
exclusively related to the female offspring. The IL6-174 CC genotype associates with young age at
We identified families with female offspring in which one onset in female T1DM offspring
parent was homozygous (CC or GG) and the other parent was The genotypes of the IL6-174G/C SNP have been shown to
heterozygous. This approach allowed us to evaluate both the affect the age of onset in other diseases (33,38). Thus, we
parental transmission and the putative effect of the non- evaluated the effect of the IL6-174G/C variant on the age at
informative (homozygous) parents genotype, albeit signifi- onset of T1DM by evaluating both the transmission pattern to
cantly reducing the number of informative meioses. In families T1DM offspring above and below the median age at onset in
with one CC-homozygous and one CG-heterozygous parent the the cohort, and by evaluating the effect of gender, genotype and
distortion of the informative parents transmissions to both interaction terms of the two (categorical, linear and recessive
T1DM (n 33) and non-T1DM (n 16) female offspring was effect of the C-allele) on the age at onset in index T1DM male
profound; 26 of the 33 informative transmissions to T1DM and female offspring by two-way ANOVA.
female offspring were IL6-174C alleles (79%; 95% CI 6593%, In the young onset T1DM offspring (<11.1 years, n 208)
Ptdt 9 " 10#4, Ptdt,c 3.6 " 10#3, corrected for n 4 compar- increased IL6-174C transmission was observed since 58% (107
isons), whereas only three of 16 informative transmissions to of 186 informative transmissions; 95% CI 5165%; Ptdt 0.04)
non-T1DM female offspring were IL6-174C alleles (19%; 95% were IL6-174C transmissions. Random transmission was
CI 038%, Ptdt 0.0124, Ptdt,c 0.05). Heterogeneity analysis observed in the high age at onset group: 64 IL6-174G and 73
of the transmission pattern between the T1DM and non-T1DM IL6-174C transmissions, respectively. However, no significant
female offspring in these families revealed highly significant difference in transmission patterns between the two age-at-onset
differences; P 2.2 " 10#4, Pc 8.8 " 10#4 (corrected for groups was found; P 0.45.
n 4 comparisons). In families with one GG homozygous In order to minimize a familial effect on the age at onset only
parent and one GC heterozygous parent the transmission the 165 index female and the 168 index male offspring were
pattern was not distorted; 29 of 49 (Ptdt 0.2) and 16 of 33 included. The mean and median ages of onset in T1DM
transmissions to T1DM and non-T1DM females, respectively, female and male offspring grouped by IL6-174G/C genotype are
were IL6-174C transmissions. Despite the low number infor- illustrated in Figure 1. The overall median age of onset in the
mative transmissions in the two groups (GG/GC versus CC/GC male and female index cases demonstrated significantly younger
parents) a borderline significant (uncorrected) difference in onset of T1DM in the female group; male versus female median;
transmission pattern to T1DM females was observed, P 0.064. 12.20 versus 9.30 years, P 2.8 " 10#3. The fitted two-way
Hence, an effect of both the genotype of the TDT non- ANOVA demonstrated an effect of gender on age at onset;
informative parent (IL6-174G protects) and a profound effect of P 1.5 " 10#3. This analysis did not show an overall effect of
the transmitted allele from the TDT informative parent were the genotype on the age at onset; P 0.43. The categorical and
observed; IL6-174C and IL6-174G confer risk and protection, linear interaction between gender and genotype on the age at
1104 Human Molecular Genetics, 2003, Vol. 12, No. 10
onset was not significant; F(2,327) 1.56, P 0.21 and and PMA-treated activities of the two different construct
F(1,328) 1.64, P 0.20, respectively. In the recessive-based variants without E2 preincubation; P 1.7 " 10#3; Fig. 3). The
model a trend for interaction of gender and genotype was PMA stimulated activity of the 225IL6C-pGL3 construct
observed [F(1,328) 3.07, P 0.08], suggesting that the exceeded the PMA stimulated activity of the 225IL6G-pGL3
C-allele in a recessive way may affect age at onset differently construct; Pc 0.038 (Fig. 3). PMA treatment did not stimulate
in males and females. In the interaction model the age at onset the promoter activity of the 225IL6G-pGL3 construct. The
was significantly different between males and females with the constitutive activities of the two construct variants displayed no
CC-genotype, (means: (males), 14.04, 95% CI 11.6216.46 and significant differences.
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 PMA stimulates the IL6-174G promoter in the presence of
gender on age at onset was clearly demonstrated by the 17b-estradiol
cumulative distribution function of age in the six genotype/
gender groups (Fig. 2). This figure clearly visualizes the A significant difference in activity between the four conditions
differences in age at onset of T1DM between the IL6-174 CC with E2 preincubation was demonstrated (P 0.048; Fig. 3).
males and females. Taken together, the above analyses suggest The PMA stimulated activities of the two construct variants
that age at onset of T1DM in females is affected by the IL6-174 displayed no significant difference; Pc 0.12.
CC genotype. Importantly, the observations also suggest that the The PMA-treated activity of the two construct variants with
difference in age at onset between boys and girls is partly and without E2 preincubation was evaluated and significant
explained by the fact that girls carrying the CC genotype had differences were demonstrated; P < 6.7 " 10#5 (Fig. 3). The
lower age of onset. main cause of this highly significant difference was the low
activity of the 225IL6G-pGL3 construct in the absence of
The IL6-174C promoter variant determines higher E2 preincubation (Fig. 3). This difference was corrected by the
stimulated but not constitutive promoter activity in the presence of E2, Pc 0.024 (Fig. 3). No differences in
Ishikawa cell line in the presence of 17b-estradiol constitutive activities between of the two construct variants with
and without E2 preincubation were observed, P 0.33 (Fig. 3).
The human Ishikawa endometrial adenocarcinoma cell line was Evaluation of the PMA stimulation indices (calculated as the
demonstrated to express the human estrogen receptor (hER) by ratio of mean PMA stimulated activity :mean constitutive
western blotting (data not shown). We observed significant activity of the construct in each experiment) of the two-
differences in activity between the four conditions (constitutive construct variant with and without E2 preincubation (Fig. 4) by
Human Molecular Genetics, 2003, Vol. 12, No. 10 1105
born approximately a mean of 30 years later than the T1DM and IL-1b stimulated promoter activity with higher stimulated
cases (population admixture?), born by normal delivery activity of the IL6-174G construct compared to IL6-174C
(selection?) and born by mothers having no familial history construct (28). This observation was challenged by the finding
of diabetes (selection?). Finally, the numbers of individuals in of marginally increased activity of the IL6-174C constructs
the casecontrol study groups were small. It is most unlikely compared with IL6-174G constructs in truncated (#211 to
that the association of T1DM with different alleles in the 13) IL6 promoter constructs (32). In light of the above studies
present and the UK casecontrol study (27) reflects oppositely and our TDT data we were prompted to investigate the
directed linkage disequilibrium (LD) with a closely linked hypothesis that the activity of the two IL6-174G/C SNP
culprit T1DM risk gene variant. TDT analysis of candidate variants was differently affected by E2. We found that the
gene variations with minor impact is more powerful than sib- inability of PMA to stimulate the IL6-174G variant was
pair-based analyses (40), and the TDT analysis does not suffer corrected by E2 preincubation whereas the PMA stimulated
from risk of spurious association as do genetic casecontrol activity IL6-174C promoter variant was unaffected by E2. We
studies using small samples (39). Further, we demonstrated did not confirm the previously reported decreased IL6 promoter
highly significant intra-familial heterogeneities in the transmis- activity by E2 in Ishikawa cells (37), which may be due to hER
sion patterns between affected males and females and between down regulation (37), or differences in assay conditions. The
affected and unaffected female offspring. SNP map to a negative regulatory domain (#224 to #158 bp)
Our observation outlines the necessity of evaluating genetic in the IL6 promoter (45). This site, however, has not been
linkage and association data in T1DM not only in analyses directly implicated in the E2 regulation of IL6 promoter activity,
grouped by HLA class II genotype or HLA class II sharing which is most likely mediated through a direct binding of
Cloning of the Luciferase-reporter constructs Expression control of the human estrogen receptor (hER) in the
human endometrial adenocarcinoma Ishikawa cell line (in which
The two allelic forms of the IL6-174G/C SNP were cloned into PMA has been shown to induce IL6 promoter activity and E2 to
the pGL3 Basic Vector (Promega, Madison, WI, USA) by 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 In order to reduce the inter-assay variation resulting from the
western blotting analysis as described previously (49). luciferase reagents, the activity of the 225IL6C-pGL3 and
225IL6G-pGL3 variants (corrected for transfection efficacy by
Cell culture the co-transfected TK-pRL) was expressed as fold activity
compared with activity of the pGL3 Basic vector.
The Ishikawa cell line (a generous gift from B. Sehested-
Hansen, Novo Nordisk A/S) was cultured in DMEM (Gibco,
Statistical analysis
Paisley, Scotland) supplemented with non-essential AA
(Gibco), sodium-bicarbonate (Gibco) 2.2 g/l final concentra- Transmission disequilibrium testing was performed using the
tion, L-glutamine (Gibco) 2 mM final concentration, penicillin Sib-TDT analysis software (50). The 95% confidence interval for
and streptomycin (Gibco) and 10% FCS (Gibco), (named transmission distortion was calculated using the method
CM1). The cell line was grown at 37( C in 5% CO2 at an initial described in (51). Heterogeneity analysis of transmission patterns
cell concentration of 2 " 106 cells/30 ml CM1 in T175 culture between groups (w2-analyses, Yates corrected when appropriate)
flasks. The culture medium was renewed bi-daily and cells were and MannWhitney analysis of median age at onset in male and
split twice weekly by standard methods. Three days prior to females were performed using the MEDSTAT software Version
transfection the cells were split, washed and transferred to a 2.1 (Astra, DK). For the age-at-onset data we fitted a two-way
steroid-free (double-charcoal stripped FCS and phenol-red free) ANOVA with gender and genotype, and three different interaction
media (CM2) consisting of DMEM without phenol red (Gibco) terms between gender and gene-dose (categorical, linear and
with 2.2 g/l Sodium bicarbonate (Gibco), 4 mM L-Glutamine recessive effect of the IL6-174C allele) using the web-based
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