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Human Molecular Genetics, 2003, Vol. 12, No. 10 1101-1110
DOI: 10.1093/hmg/ddg132
© 2003 Oxford University Press

Association of a functional 17ß-estradiol sensitive IL6-174G/C promoter polymorphism with early-onset type 1 diabetes in females

Ole P. Kristiansen1,{dagger}, Runa L. Nolsøe1,{dagger}, Lykke Larsen1, Anette M.P. Gjesing1, Jesper Johannesen1, Zenia M. Larsen1, Anne E. Lykkesfeldt2, Allan E. Karlsen1, Flemming Pociot1, Thomas Mandrup-Poulsen1,3,*, DIEGG{ddagger} and DSGD§

1Steno Diabetes Center, DK-2820 Gentofte, Denmark, 2Department of Tumor Endocrinology, Institute of Cancer Biology, Danish Cancer Society, DK-2100 Copenhagen Ø, Denmark and 3Department of Molecular Medicine, Karolinska Institute, SE-17176 Stockholm, Sweden

Received December 16, 2002; Accepted March 10, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL
 METHODS
 REFERENCES
 
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=6.5x10-4 and Ptdt=2.4x10-4, 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=4.6x10-3, and demonstrated differences in the transmission patterns between female and male T1DM offspring, P=5.1x10-3. The IL6-174 CC genotype was associated with younger age at onset of T1DM in females (P=0.002). The impact of 17ß-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
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIAL
 METHODS
 REFERENCES
 
Human genome-wide scans in type 1 (insulin-dependent) diabetes mellitus (T1DM) have provided evidence for T1DM as a polygenic disease (1–6), but hitherto these scans have not identified novel etiological T1DM gene variants. T1DM is considered to be an autoimmune disease (7). Thus, polymorphisms in genes encoding proteins with known or inferred impact on immunity and autoimmunity may be considered candidate gene variants for T1DM risk.

Interleukin-6 (IL-6) is a pleiotropic cytokine known to be 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 hypothalamic–pituitary–adrenal 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 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 ß-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 (20–22). 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 (1–6) 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 3' region of IL6. Recently, a case–control study in UK type 1 diabetics demonstrated 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 (28–30) 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 (28,33–35). Finally, the activity of the IL6 promoter has been shown to be suppressed by 17ß-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
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIAL
 METHODS
 REFERENCES
 
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 Hardy–Weinberg 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 unaffected offspring, Table 1.


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  		Table 1. Transmission of IL6-174G/C alleles in Danish T1DM families
 
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 offspring, excluding a putative transmission distortion ratio for this variation in all female offspring (Table 2). The transmissions 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=1x10-3 (Table 2). This pattern was not found in the 168 male index cases (defined as above for the T1DM female offspring; Table 2).


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  		Table 2. Transmission of IL6-174G/C alleles in female and male offspring
 
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 differences between the two groups (all cases, P=5.1x10-3, Pc=0.02; and index-cases, P=5.7x10-3, 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) parent's genotype, albeit significantly 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 65–93%, Ptdt=9x10-4, Ptdt,c=3.6x10-3, corrected for n=4 comparisons), whereas only three of 16 informative transmissions to non-T1DM female offspring were IL6-174C alleles (19%; 95% CI 0–38%, 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=2.2x10-4, Pc=8.8x10-4 (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 informative 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 51–65%; 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=2.8x10-3. The fitted two-way ANOVA demonstrated an effect of gender on age at onset; P=1.5x10-3. 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 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 11.62–16.46 and females, 8.81, 95% CI 6.60–11.01; 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.



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  		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: females—CC (n=47), CG (n=76) and GG (n=42), respectively; males—CC (n=39), CG (n=77) and GG (n=52), respectively.

 


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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: females—CC (n=47), CG (n=76) and GG (n=42), respectively; males—CC (n=39), CG (n=77) and GG (n=52), respectively.

 
The IL6-174C promoter variant determines higher stimulated but not constitutive promoter activity in the Ishikawa cell line in the presence of 17ß-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 and PMA-treated activities of the two different construct variants without E2 preincubation; P=1.7x10-3; 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.



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  		Figure 3. Constitutive and PMA stimulated activity of 225IL6G/C-pGL3 constructs transfected into Ishikawa cells grown with and without 17ß-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.

 
PMA stimulates the IL6-174G promoter in the presence of 17ß-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<6.7x10-5 (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 ANOVA revealed significant differences between the conditions; 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 stimulation index of the 225IL6G-pGL3 construct stimulation index without of E2 present (Fig. 4).



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  		Figure 4. Stimulation indices of 225IL6G/C-pGL3 constructs transfected into Ishikawa cells grown with and without 17ß-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.

 
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 1003–1350) 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
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIAL
 METHODS
 REFERENCES
 
We demonstrate evidence for significant linkage in the presence of association between the IL6-174G/C SNP and T1DM in a 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 demonstrated. 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 female–female 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 32–58%. This is not different from the IL6-174C transmission (95% CI 51–61%) 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 79–89% 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, 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 case–control study groups were small. It is most unlikely that the association of T1DM with different alleles in the present and the UK case–control 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 case–control studies using small samples (39). Further, we demonstrated highly significant intra-familial heterogeneities in the transmission 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 may—at least in part—be 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 3'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 3'-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 association 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-1ß 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 NF-IL6 and NF-{kappa}B 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 5' 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 availability 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 development, 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
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL
METHODS
 REFERENCES
 
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 0–45), respectively. All families were unrelated.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL
 METHODS
REFERENCES
 
Genotyping for the IL6-174G/C polymorphism
This was performed by a PCR based RFLP assay. 164 bp (position 939–1102, GenBank accession no. Y00081.1; -222 to -59 relative to the transcription start site) (47) of the IL6 promoter were amplified using the primers 5'-GCC TCA ATG ACG ACC TAA GC-3' and 5'-TCA TGG GAA AAT CCC ACA TT-3'. The PCR mixture comprised 50 µM of each dNTP (Life Technologies, Paisley, UK), 1.5 mM MgCl2, 0.5 U Taq-polymerase (Life Technologies), 1 µM of each primer and 1xpolymerase buffer (Life Technologies) and 40 ng genomic DNA in total reaction volume of 20 µl. 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 µg 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 1003–1350 ) 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 5'-TTC CCA TCA AGA CAT GCT CA-3' (forward) and 5'-GCA AGG AAC TGC CTT CAC TTA-3'. 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 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 5'-CCT GCA AGA GAC ACC ATC CT-3' (forward) and 5'-TCC TGG AGG GGA GAT AGA GC-3' (reverse) amplifying the -1153 to +54 region GenBank accession no. Y00081; 8–1214) of the IL6 promoter. This was followed by nested PCR using the primers 5'-GGG GTA CCC CGA CAC CAT CCT GAG GGA AGA (forward) and 5'-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 5' and 3' ends, respectively, in an amplicon comprising the -1144 to +24 bp region of the promoter (GenBank accession no. Y00081; 17–1184). Restriction cleavage of the amplicons and the pGL-3 Basic Vector using the KpnI and HindIII (both from New England Biolabs) restriction enzymes was performed. The cleaved products were ligated directly into the similarly cleaved and dephosphorylated pGL3 Basic Vector using the Original TA cloning® kit (Invitrogen, Carlsbad, CA, USA). Promoter positive clones were identified by use of the primers 5'-GCC AGA ACA TTT CTC TAT CG-3' (forward, denoted 5200) and 5'-TAC CAA CAG TAC CGG AAT GC-3' (reverse, denoted 5201). Promoter positive constructs were transfected into One-Shot® 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 manufacturer's 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 manufacturer's 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 20–40% without 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 concentration, 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 2x106 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 ß-D-glucose (total concentration in medium 4.5 g/l, Sigma) and 5% double-charcoal stripped FCS (Gibco) at an initial cell concentration 2x106 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 µl CM2/well). On the day of transfection the medium was removed and replaced by 400 µl 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 µl SuperfectTM (Qiagen) in a total volume of 100 µl were incubated for 15 min at room temperature according to the manufacturer's 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 µl 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 manufacturer's 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 ({chi}2-analyses, Yates' corrected when appropriate) and Mann–Whitney 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 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.


   FOOTNOTES
 
* 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{at}steno.dk Back

{dagger} The authors wish it to be known that, in their opinion, the first two should be regarded as joint First Authors. Back

{ddagger} The Danish IDDM Epidemiology and Genetics Group. Back

§ The Danish Study Group of IDDM in Childhood. Back


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