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Is Puberty an Accelerator of Type 1 Diabetes in IL6-174CC Females?

¹ Department of Diabetes and Metabolism, Division of Medicine, University of Bristol, U.K
² Steno Diabetes Center, Gentofte, Denmark
³ Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden

	ABSTRACT

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The pubertal peak in onset of type 1 diabetes occurs earlier in girls than boys. We postulated that this sex difference might be mediated in part by estrogen or by genes regulated by estrogen, such as the interleukin-6 (IL6) gene. Previous studies concerning the role of an estrogen-sensitive single nucleotide polymorphism (SNP) in the IL6 promoter in type 1 diabetes have proved contradictory. We therefore selected a large, genetically homogenous population-based cohort, analyzed by age at onset and sex, to test the hypothesis that the IL6-174G>C SNP affects age at onset of type 1 diabetes in females but not in males. We found that the IL6-174CC genotype was significantly less frequent in females diagnosed after than in those diagnosed before the age of 10 years (19 vs. 13%, P = 0.016). No genotype difference was observed in males stratified for age at onset. Among children diagnosed after age 10, the median age of onset was 11.9 years (intraquartile range 10.7–14.6) in 34 girls homozygous for IL6-174C compared with 13.2 years (11.6–15.4) in 229 girls with other genotypes and 13.5 years (12.0–15.6) in 339 males with any IL6-174 genotype (P = 0.012). These data support the hypothesis that pubertal changes may contribute to accelerated onset of type 1 diabetes in genetically susceptible females. This phenomenon may be orchestrated by the action of estrogen on the IL6 promoter.

IL6 is a pleiotropic cytokine expressed by a wide variety of cells: T- and B-cells, macrophages, dendritic cells, fibroblasts, and endothelial and pancreatic ß-cells. A GC polymorphism has been reported at position –174 of the IL6 promoter affecting IL6 transcription (3). A U.K. case-control study (4) found that the homozygous IL6-174G/G genotype was increased in patients with type 1 diabetes compared with healthy control subjects (P < 0.002), with a concomitant decrease in the IL6-174C/C genotype in the patients compared with control subjects (12.5 vs. 24.2%, respectively, P < 0.004) (4). In the 53 trios studied, the IL6-174G allele was transmitted in 29 of 53 (54.7%) informative meioses, and there was no association with age at onset. In contrast, in 206 Dutch simplex families where the proband was diagnosed under the age of 14 years, no association with the IL6-174G>C polymorphism was identified (5). A recent study of 253 Danish affected sibpair and simplex families (6) showed that the IL6-174C allele was strongly associated with type 1 diabetes in females but not in males and showed an age at onset effect in females. In this study, an increased transmission of 66% (95% CI 0.58–0.74) of the IL6-174C allele to females was observed.

The apparently discrepant results from these studies (4–6) may, however, be due to lack of power, to differences in selection criteria, or to differences between populations. The most rigorous test of association of a single nucleotide polymorphism (SNP) with a disease phenotype is through analysis of large homogeneous population-based cohorts (7). We therefore set out to test the hypothesis that there is a sex difference in the frequency of an estrogen-sensitive SNP in 1,130 individuals diagnosed with type 1 diabetes before the age of 21 years and participating in a population-based cohort from the U.K. (8). This study did not require control data, since it focused simply on whether there is a sex- and age-related difference in the frequency of a functional polymorphism in a genetically homogenous cohort with ages at diagnosis covering the onset of puberty.

Of the 1,130 individuals studied, 609 were male. Analysis of the frequency of the IL6-174G>C polymorphism demonstrated that the IL6-174G>C polymorphism was in Hardy-Weinberg equilibrium (P = 0.78), and no overall significant difference (P = 0.27) according to sex was observed in the overall IL6-174G>C genotype distributions (Table 1A). When males and females diagnosed over the age of 10 years were analyzed, there was, however, a difference in the frequency of the IL6-174G>C polymorphism between the two groups (genotype-wise P = 0.02 and allele-wise P = 0.05) (Table 1B). This difference was not detected in boys and girls diagnosed under the age of 10 years (Table 1C). The frequency of the IL6-174G>C polymorphism in girls diagnosed after the age of 10 was significantly lower compared with girls diagnosed earlier and compared with all males regardless of age (13 vs. 19% P = 0.016).

View larger version (51K): [in this window] [in a new window]   FIG. 1. The cumulative age of onset in 1,130 individuals with type 1 diabetes stratified by each IL6-174G>C genotype and sex.

This study, designed to examine the role of an estrogen-sensitive polymorphism in the IL6 promoter in a population-based cohort with type 1 diabetes, showed that the frequency of the IL6-174CC genotype decreases with increasing age of onset in females but not males with type 1 diabetes. Twice as many females as males homozygous for the IL6-174C allele develop diabetes between the ages of 8.5 and 11.5 years. These data are in agreement with a recent Danish study demonstrating association between the IL6-174C allele and type 1 diabetes in females but not males in a Danish population (6) but are contrary to other studies of the IL6 gene in type 1 diabetes. It is worth noting that in the two studies (4,5) that did not find an association between IL6-174G>C and type 1 diabetes, the age at diagnosis was defined as <14 years, with a mean age of onset of 4 years in one study (5), and in the other (4), the data were not analyzed according to sex. It is possible that this type of sex effect, which is visible during adolescence, might also have been missed by whole-genome screens that usually focus on those diagnosed early in life (9,10).

Both the Danish and UK-BOX (Bart’s Oxford) populations showed that females homozygous for IL6-174CC had a younger age of onset than females with an IL6-174G allele and all males studied. There were, however, interesting differences in the results obtained from both studies. When the Danish data were corrected for age at diagnosis <21 years, the median age of onset in IL6-174CC females was 6.4 years compared with 9.2 years in the BOX cohort. Furthermore, the cumulative incidence of type 1 diabetes, stratified according to age at onset and genotype, is linear in the U.K. population until the age of 8.5–9.5 years (Fig. 1), after which IL6-174CC females appear to have a left shift in age of onset of disease. In the Danish population, IL6-174CC females have an earlier age of onset throughout life (6).

We postulate that differences in the two populations tested may be responsible for the stronger effect of the IL6(-174)CC genotype in the Danish study. The cohort of 253 families comprised 150 affected sibpair families and 103 parent/offspring families collected retrospectively through adult and pediatric clinics. The families participating in the Bart’s Oxford population-based study were identified prospectively with 95% case ascertainment (8); only 10% are affected sibpair families. Affected sibpair and affected parent/offspring families may be enriched for diabetes susceptibility genes, resulting in a greater effect in these families. We also considered that Denmark may be a higher-risk population than the U.K., but recent epidemiological data indicate that the incidence of type 1 diabetes is similar in both populations (11).

The mechanism by which the IL6-174CC genotype increases risk of type 1 diabetes in females but not in males is unclear. One possible explanation of the U.K. cumulative incidence stratified according to age at onset and genotype (Fig. 1) is that estrogen, switched on from 9 years of age onwards (12) in females, has increased risk in IL6-174CC females, resulting in earlier age of onset. An effect of oral contraceptives in young women may be hidden within the 15- to 20-year-old group, as it has been reported that in some women, use of oral contraceptives affects IL6 production (13).

Previous reporter studies have confirmed that estrogen plays a role in IL6 regulation, although some of these data remain controversial (2,3,6). The first 225 bp of the promoter is sufficient for synergistic activation of IL6 by the transcription factors nuclear factor (NF)-IL6 and NF-B, and estrogen (E₂) receptors can interact directly with the NF-IL6/NF-B complex, inhibiting their ability to bind to the IL6 promoter and activate gene transcription (14). The IL6-174GC SNP lies adjacent to the multiple response binding site for NF-IL6, but its effect has proven contentious. An initial study using reporter assays showed that the IL6-174 G allele was more active than the IL6-174C allele in stimulated HeLa cells (3). A later study presented data indicating that the IL6-174C allele may be more active than the G allele in HeLa cells, but these differences did not reach statistical significance. The study emphasized that genetic regulation of IL6 is controlled by several promoter variants and is cell specific (15).

The effect of the IL6-174G>C polymorphism has recently been analyzed in Ishikawa cells expressing the human estrogen receptor to elucidate the role of estrogen on the two different alleles of the IL6-174G>C polymorphism (6). The two alleles responded differently to E₂ pretreatment; when stimulated in the absence of estrogen, the IL6-174C allele was 70% more active than the IL6-174G allele, and in the presence of estrogen, the activity of the IL6-174G allele was increased so that both alleles have equal function. These results do not offer an explanation of our genetic data, but it is possible, indeed likely, that the regulation of IL6 in lymphocytes and islet ß-cells differs from its regulation in the cell types previously studied. Further studies of the genetic regulation of IL6 will be required to explain the mechanism underlying the increased risk to IL6-174CC females at puberty demonstrated in this study.

In conclusion, we have used a rigorous strategy to test the hypothesis that there is a sex difference in the frequency of an estrogen-sensitive SNP in individuals with type 1 diabetes. Our data demonstrate a role for the IL6-174CC genotype in the susceptibility of adolescent females to type 1 diabetes and provide preliminary evidence for a role for an estrogen-sensitive gene in determining the observed sex difference in type 1 diabetes susceptibility at puberty. On the basis of these data, we propose that haplotypes of IL6 in combination with alleles of other genes regulated by estrogen may provide the key to the sex differences observed in type 1 diabetes.

	RESEARCH DESIGN AND METHODS

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DNA was collected from the probands of families participating in the BOX Study of Childhood Diabetes. This is a prospective, population-based family study that has, since 1985, recruited >95% of the families of children who have developed type 1 diabetes before age 21 years in the former Oxford Health Authority Region, U.K. (8). By March 2002, 1,746 families had been recruited into the study, with 89% under regular follow-up. The study population is 95% white European, and the remainder originate mainly from the Indian subcontinent. The classification of type 1 diabetes was based on assignment by the referring clinician and was made on the basis of World Health Organization criteria (16) and a clinical requirement for insulin treatment from diagnosis. Patients with secondary diabetes, known genetic subtypes including MODY (maturity-onset diabetes of the young), or clinical type 2 diabetes were not included in the study. The study was approved by the research ethics committees in all participating centers.

Genetic analysis.
DNA extraction and HLA class II genotyping were performed as previously described (17). The IL6-174G>C SNP was analyzed in 1,130 individuals (609 male) with type 1 diabetes using PCR-RFLP (restriction fragment–length polymorphism) as described previously (6).

Statistical analysis.
Frequency differences in the IL6 polymorphism between males and females were analyzed overall and according to age at diagnosis using the ² and Fisher’s exact tests. For the interaction between age at onset and genotype, a two-way ANOVA was used. Comparison of median age at diagnosis in all groups was analyzed by Kruskal-Wallis testing. All data were stored according to existing data protection regulations within the department.

	ACKNOWLEDGMENTS

 
This study was funded by Diabetes U.K., The Wellcome Trust, and Novo Nordisk.

We acknowledge the continuing help of the children participating in the BOX study and their families, as well as the field workers and the administrative team. We are very grateful to Marja Deckert, Kyla Chandler, and Rachel Dix for technical assistance and to Bendix Carsten for help with the statistical analysis.

	FOOTNOTES

 
T.M.-P. is employed by, holds stock in, and has received grant/research support from Novo Nordisk.

Address correspondence and reprint requests to Dr. K.M. Gillespie, Medical School Unit, Southmead Hospital, Bristol BS10 5NB, U.K. E-mail: k.m.gillespie{at}bristol.ac.uk

Received for publication October 7, 2004 and accepted in revised form December 16, 2004

Abbreviations: BOX, Bart’s Oxford; IL, interleukin; NF, nuclear factor; SNP, single nucleotide polymorphism

	REFERENCES

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