Dynamic demethylation of the IL2RA promoter during in vitro CD4+ T cell activation in association with IL2RA expression.

IL2RA, a subunit of the high affinity receptor for interleukin-2 (IL2), plays a crucial role in immune homeostasis. Notably, IL2RA expression is induced in CD4+ T cells in response to various stimuli and is constitutive in regulatory T cells (Tregs). We selected for our study 18 CpGs located within cognate regulatory regions of the IL2RA locus and characterized their methylation in naive, regulatory, and memory CD4+ T cells. We found that 5/18 CpGs (notably CpG + 3502) show dynamic, active demethylation during the in vitro activation of naive CD4+ T cells. Demethylation of these CpGs correlates with appearance of IL2RA protein at the cell surface. We found no influence of cis located SNP alleles upon CpG methylation. Treg cells show constitutive demethylation at all studied CpGs. Methylation of 9/18 CpGs, including CpG +3502, decreases with age. Our data thus identify CpG +3502 and a few other CpGs at the IL2RA locus as coordinated epigenetic regulators of IL2RA expression in CD4+ T cells. This may contribute to unravel how the IL2RA locus can be involved in immune physiology and pathology.

Impact of social inequalities at birth on the longevity of children born 1914-1916: A cohort study.

BACKGROUND: Testing whether familial socioeconomic status (SES) in childhood is a predictor of mortality has rarely been done on historical cohorts. METHODS: The birth certificates of 4,805 individuals born 1914-1916 in 16 districts of the Paris region were retrieved. The handwritten information provided the occupation of parents, the legitimacy status, life events (e.g. marriage, divorce), and the precise date of death when after 1945 (i.e. age 31 years (y) in the cohort). We used the median age at death (MAD) as a global measure of mortality, then studied separately survival to and after 31 y. Multivariate Imputation by Chained Equations (MICE), Generalized Additive Models (GAMs) and mixed effect Cox models were used. RESULTS: MAD showed large variations according to paternal occupation. The lowest MAD in both sexes was that of workers' children: it was 56.3 y (95% CI: [48.6-62.7]) in men and 67.4 y (95% CI: [60.8-72.7]) in women, respectively (95% CI: 13.4 y [5.7-21.3]) and 12.3 y (95% CI: [4.0-19.2]) below the highest MAD attained. MAD experienced by illegitimate children was 18.9 y (95% CI: [13.3-32.3]) shorter than of legitimate children. The multivariate analysis revealed that in both sexes survival to age 31 y was predicted independently by legitimacy and paternal occupation. Paternal occupation was found significantly associated with mortality after age 31 y in females only: accordingly difference in life expectancy at age 31 y was 4.4 y (95% CI: [1.2-7.6]) between upper class and workers' daughters. CONCLUSIONS: Paternal occupation and legitimacy status were strong predictors of offspring longevity in this one-century historical cohort born during World War One.

Using spatio-temporal surveillance data to test the infectious environment of children before type 1 diabetes diagnosis.

The "hygiene hypothesis" postulates that reduced exposure to infections favours the development of autoimmunity and childhood type 1 diabetes (T1D). But on the other side, viruses, notably enteroviruses, are suspected to trigger T1D. The assessment of the possible relationships between infections and T1D still defies the classical tools of epidemiology. We report the methods and results of a geographical approach that maps the addresses of patients to a communicable diseases surveillance database. We mapped the addresses of patients at birth, infancy and T1D diagnosis to the weekly estimates of the regional incidences of 5 frequent communicable diseases routinely collected since 1984 by the French Sentinel network. The pre-diagnostic infectious environment of 3548 patients with T1D diagnosed between 0.5 and 15 years was compared to those of 100 series of age-matched "virtual controls" drawn randomly on the map. Associations were classified as "suggestive" (summer diarrhea, SD, and varicella, V) when p< 0.05, or "significant" (influenza-like infections, ILI) when they passed the Bonferroni correction for FDR. Exposure to ILI and SD were associated with T1D risk, while V seemed protective. In the subset of 2521 patients for which we had genome wide data, we used a case-only approach to search for interactions between SNPs and the infectious environment as defined by the Sentinel database. Two SNPs, rs116624278 and rs77232854, showed significant interaction with exposure to V between 1 and 3 years of life. The infectious associations found should be taken as possible markers of patients' environment, not as direct causative factors of T1D. They require replication in other populations. The increasing public availability of geographical environmental databases will expand the present approach to map thousands of environmental factors to the lifeline of patients affected by various diseases.

Comparison of a French pediatric type 1 diabetes cohort's responders and non-responders to an environmental questionnaire.

BACKGROUND: Type 1 diabetes (T1D) incidence has doubled since the 1980's for children aged <5 years old, potentially relevant environmental factors having thus to be sought early in the patient's life. The identification of environmental factors that can explain the changing epidemiology of T1D requires comprehensive environmental inquiries. However, a limitation is the willingness of patients and families to complete these environmental questionnaires. Our objective was to identify patients' personal and social characteristics predictive of the return, time to the return and completeness of a comprehensive environmental questionnaire. METHODS: The parents of 2832 T1D patients aged <15 years old enrolled in the French Isis cohort were sent a 1379-item environmental questionnaire. A geographic information system was used to collect information on patients' socioeconomic environment. Multivariate statistical analyses were conducted to identify predictors of questionnaire return, time to its return and its completeness. RESULTS: Within 6 months, 867 (30.6%) questionnaires were returned. Socioeconomic environment was strongly associated with the probability of response, with fewer responses from cities with high Townsend deprivation index (p =2 × 10-7), high unemployment (p =0.005), blue-collar workers' rate (p =0.0002) and household overcrowding (p =0.02). Response rates were similar for male and female patients, but were higher for less severely affected patients (p =0.006) and younger patients (p =5 × 10-5). When returned, completeness was high with a mean of 96%. CONCLUSION: Identification of personal or socioeconomic characteristics differing between questionnaire responders and non-responders may help target future environmental investigations on those patients who will more likely return the information, and reduce bias using these variables to stratify the analyses.

CpG methylation changes within the IL2RA promoter in type 1 diabetes of childhood onset.

None of the polymorphic variants of the IL2RA gene found associated with Type 1 Diabetes (T1D) was shown to have a functional effect. To test if the epigenetic variation could play a role at this locus, we studied the methylation of 6 CpGs located within the proximal promoter of IL2RA gene in 252 T1D patients compared with 286 age-matched controls. We found that DNA methylation at CpGs -373 and -456 was slightly but significantly higher in patients than in controls (40.4 ± 4.6 vs 38.3 ± 5.4, p=1.4E4; 91.4 ± 2.8 vs 89.5 ± 5.3, p=1.8E-6), while other CpG showed a strictly comparable methylation. Among 106 single nucleotide polymorphisms (SNPs) located in the neighboring 180 kb region, we found that 28 SNPs were associated with DNA methylation at CpG -373. Sixteen of these SNPs were known to be associated with T1D. Our findings suggest that the effect of IL2RA risk alleles on T1D may be partially mediated through epigenetic changes.

Association of the CpG methylation pattern of the proximal insulin gene promoter with type 1 diabetes.

The insulin (INS) region is the second most important locus associated with Type 1 Diabetes (T1D). The study of the DNA methylation pattern of the 7 CpGs proximal to the TSS in the INS gene promoter revealed that T1D patients have a lower level of methylation of CpG -19, -135 and -234 (p = 2.10(-16)) and a higher methylation of CpG -180 than controls, while methylation was comparable for CpG -69, -102, -206. The magnitude of the hypomethylation relative to a control population was 8-15% of the corresponding levels in controls and was correlated in CpGs -19 and -135 (r = 0.77) and CpG -135 and -234 (r = 0.65). 70/485 (14%) of T1D patients had a simultaneous decrease in methylation of CpG -19, -135, -234 versus none in 317 controls. CpG methylation did not correlate with glycated hemoglobin or with T1D duration. The methylation of CpG -69, -102, -180, -206, but not CpG -19, -135, -234 was strongly influenced by the cis-genotype at rs689, a SNP known to show a strong association with T1D. We hypothesize that part of this genetic association could in fact be mediated at the statistical and functional level by the underlying changes in neighboring CpG methylation. Our observation of a CpG-specific, locus-specific methylation pattern, although it can provide an epigenetic biomarker of a multifactorial disease, does not indicate whether the reported epigenetic pattern preexists or follows the establishment of T1D. To explore the effect of chronic hyperglycemia on CpG methylation, we studied non obese patients with type 2 diabetes (T2D) who were found to have decreased CpG-19 methylation versus age-matched controls, similar to T1D (p = 2.10(-6)) but increased CpG-234 methylation (p = 5.10(-8)), the opposite of T1D. The causality and natural history of the different epigenetic changes associated with T1D or T2D remain to be determined.

Heterogeneity of class I INS VNTR allele association with insulin secretion in obese children.

On the basis of the near-complete linkage disequilibrium of the insulin variable number of tandem repeats (INS VNTR) allele with the neighboring -23Hph1 A/T single-nucleotide polymorphism, previous studies have documented the association of class I ("short") and class III ("long") INS VNTR alleles with metabolic parameters, including circulating insulin levels. Using a new method to sequence class I alleles, we revisited this association in 346 obese children. Class I alleles are made of several types of repeats, whose repartition determines subclasses IC and ID. Fasting insulin was found to be higher in obese children with ID/ID genotypes (135 +/- 12 pmol/l, n = 64) than with ID/IC or IC/IC genotypes (91 +/- 5 pmol/l, n = 97, P = 0.0005). In response to oral glucose, peak insulin levels and insulin-to-glucose area under the curve ratios were higher in ID/ID (872 +/- 122 pmol/l and 109 +/- 15, respectively) than in ID/IC or IC/IC patients (586 +/- 42 pmol/l and 76 +/- 5, P = 0.02 and P = 0.04, respectively). Fasting and postglucose insulin levels were comparable in carriers of IC and of class III alleles. Our results support that the molecular structure of the VNTR allele, not only its overall length, is associated with variations of insulin secretion. ID/ID homozygosity appears responsible for the increased insulin levels previously attributed to the whole class I VNTR group. It will be important to test the ramifications of this observation for class I association with Type 1 (susceptibility) and Type 2 diabetes (protection).

Association of Kir6.2 and INS VNTR variants with glucose homeostasis in young obese.

Although insulin secretion is commonly increased and glucose tolerance decreased in young obese patients, there is a wide individual variability of these parameters. We investigated whether common variants at the Kir6.2 (KCNJ11) and insulin variable number of tandem repeat (INS VNTR) loci are associated with insulin or glucose levels in 388 obese children. The E23K and INS VNTR alleles showed no significant association when each locus was examined individually but a clear effect when the two loci were combined for analysis. In obese children with Kir6.2 KK and class III VNTR alleles, fasting glucose was slightly but consistently greater (4.76 +/- 0.05 mM) than in those with Kir6.2 EE and class I/I VNTR alleles (4.63 +/- 0.06 mM, P = 6.10(-4)) or other genotypes (4.64 +/- 0.03 mM, P = 1.10(-3)). Obese children with KK and class III VNTR genotypes also had an early response to oral glucose diminished by approximately 36% [insulinogenic index (IGI) = 50 +/- 4] compared with Kir6.2 EE and class I/I (IGI = 78 +/- 7, P = 0.026) or other genotypes (IGI = 69 +/- 3, P = 0.001). In young European obese, the polymorphisms of Kir6.2 and INS VNTR are thus associated with a trend for lower insulin and higher glucose levels, which may reveal a possible epistatic genetic effect that may influence a prediabetic trait in young obese children.

The common -866 G/A polymorphism in the promoter of uncoupling protein 2 is associated with increased carbohydrate and decreased lipid oxidation in juvenile obesity.

Uncoupling protein (UCP) 2 is a member of the mitochondrial transporter superfamily that uncouples proton entry in the mitochondrial matrix from ATP synthesis. Although its physiological role remains to be established, UCP2 is considered a candidate gene for association with energy metabolism and obesity. A common promoter polymorphism, -866 G/A, has been associated with increased UCP2 gene expression and middle-aged adult obesity. In fact, our analysis of 296 juvenile obese and 568 nonobese control subjects revealed no difference in the prevalence of this polymorphism. Insulin and glucose response to oral glucose was comparable across the -866 genotypes. Metabolic studies in 147 of these juvenile obese subjects showed that homozygosity for the UCP2 promoter variant A was associated with important changes in energy metabolism compared with other genotypes, i.e., a 34% increase of carbohydrate oxidation (94 +/- 10 vs. 70 +/- 3 mg.min(-1).m(-2), P = 0.004) and a 23% decrease of lipid oxidation (26 +/- 3 vs. 34 +/- 1 mg.min(-1).m(-2), P = 0.03). Therefore, the juvenile obese subjects who are homozygous for the A variant have an increased ratio (3.6 +/- 1.2) of calories derived from carbohydrates to those from lipids compared with G/A or G/G obese children (1.4 +/- 0.2, P = 0.003), suggesting a role for UCP2 in the partitioning of metabolic fuels.

Increased insulin resistance in obese children who have both 972 IRS-1 and 1057 IRS-2 polymorphisms.

In two cohorts of 174 and 165 obese Caucasian children, we measured insulin sensitivity and genotyped insulin receptor substrate IRS-1 and IRS-2 genes for the Arg972Gly and the Asp1057Gly variants, respectively. Because IRS-1 and IRS-2 have complementary roles in insulin signaling, we classified the genotypes in three categories: those with none of the variants in IRS-1 or IRS-2, those with one variant in IRS-1 or IRS-2, and those with variants in both IRS-1 and 2 proteins. The obese children with either the IRS-1 or IRS-2 variant had a mean insulin sensitivity index (2.9 +/- 0.2 in cohort 1, 2.7 +/- 0.1 in cohort 2) only slightly lower than the children having no variant in either gene (3.1 +/- 0.2 and 3.5 +/- 0.3, respectively). However, patients having variant alleles in both IRS-1 and IRS-2 genes showed a 25-35% decrease in sensitivity (2.3 +/- 0.2 and 2.0 +/- 0.2, respectively) when compared with nonvariant homozygotes (P < 0.001). These observations are reminiscent of the insulin sensitivity phenotypes in double IRS-1(+/-) IRS-2(+/-) heterozygous knockout mice. Our results stress the need for combined genotype analysis when candidate genes are functionally involved in the same pathway.