Allele-specific methylation of type 1 diabetes susceptibility genes.

The susceptibility to autoimmune diseases is influenced by genes encoding major histocompatibility complex (MHC) proteins. By examining the epigenetic methylation maps of cord blood samples, we found marked differences in the methylation status of CpG sites within the MHC genes (cis-metQTLs) between carriers of the type 1 diabetes risk haplotypes HLA-DRB1*03-DQA1*0501-DQB1*0201 (DR3-DQ2) and HLA-DRB1*04-DQA1*0301-DQB1*0302 (DR4-DQ8). These differences were found in children and adults, and were accompanied by reduced HLA-DR protein expression in immune cells with the HLA-DR3-DQ2 haplotype. Extensive cis-metQTLs were identified in all 45 immune and non-immune type 1 diabetes susceptibility genes analyzed in this study. We observed and validated a novel association between the methylation status of CpG sites within the LDHC gene and the development of insulin autoantibodies in early childhood in children who are carriers of the highest type 1 diabetes risk genotype. Functionally relevant epigenetic changes in susceptibility genes may represent therapeutic targets for type 1 diabetes.

Thymus Growth and Fetal Immune Responses in Diabetic Pregnancies.

Type 1 diabetes (T1D) during pregnancy possibly affects the development of the thymus and the maturation of the immune system in the offspring. The aim of the ImmunDiabRisk study was to investigate thymus growth and maternal and fetal immune responses in pregnancies with and without T1D. The thymus circumferences of the fetuses of pregnant women with T1D (n=49) and without diabetes (n=59) were measured using ultrasound around the 29th gestational week and standardized for gestational age. Simultaneously, the frequencies and total numbers of cell markers were analyzed by flow cytometry in maternal peripheral blood, and at birth in umbilical cord blood. The standardized circumference of the thymus was similar in fetuses of mothers with and without T1D (p=0.26). We observed higher numbers of FOXP3 Tregs, memory Tregs, erythrocytes, and lymphocytes in the cord blood from T1D pregnancies (p=0.01, p=0.002, p=0.002 and p=0.02, respectively). The frequencies of CD4+/CD8+ T cells correlated positively in maternal blood and umbilical cord blood of mother-child pairs, as did the levels of neutrophils (Spearman's correlation coefficient r=0.43, p=0.02 for CD4+/CD8+ cells; r=0.46, p=0.03 for neutrophils), while no significant correlations were observed between thymus circumference and any cell markers in the child. Parts of the prenatal immune system seem to develop differently in the offspring of mothers with and without T1D. The correlation of Tregs between maternal blood and cord blood may indicate a significant cross-talk between the maternal and fetal immune system.

Human placental transcriptome shows sexually dimorphic gene expression and responsiveness to maternal dietary n-3 long-chain polyunsaturated fatty acid intervention during pregnancy.

BACKGROUND: Previously we have examined the effect of maternal dietary n-3 long-chain polyunsaturated fatty acid (LCPUFA) supplementation during pregnancy on offspring fat mass. Considering the involvement of the placenta in fetal programming, we aimed to analyze the sex-specific gene expression in human term placenta and its response to the n-3 LCPUFA intervention, as well as their correlations to offspring adiposity. RESULTS: Placental gene expression was assessed in a control and n-3 LCPUFA intervention group by DNA microarrays, biological pathway analyses and RT-qPCR validation. Expression data were correlated with sex steroid hormone levels in placenta and cord plasma, and offspring anthropometric data. Transcriptome data revealed sexually dimorphic gene expression in control placentas per se, whereas in intervention placentas sex-specific expression changed, and more n-3 LCPUFA-regulated genes were found in female than male placentas. Sexually dimorphic gene expression and n-3 LCPUFA-responsive genes were enriched in the pathway for cell cycle and its associated modulator pathways. Significant mRNA expression changes for CDK6, PCNA, and TGFB1 were confirmed by RT-qPCR. CDK6 and PCNA mRNA levels correlated with offspring birth weight and birth weight percentiles. Significantly reduced placental estradiol-17ß/testosterone ratio upon intervention found in female offspring correlated with mRNA levels for the 'Wnt signaling' genes DVL1 and LRP6. CONCLUSIONS: Overall, human placentas show sexually dimorphic gene expression and responsiveness to maternal n-3 LCPUFA intervention during pregnancy with more pronounced effects in female placentas. The absence of correlations of analyzed placental gene expression with offspring adipose tissue growth in the first year is not mutually exclusive with programming effects, which may manifest later in life, or in other physiological processes.

Gene expression profiles in placenta and their association with anesthesia, delivery mode and maternal diabetes.

INTRODUCTION: Fetal development is dependent on placenta and affected by multiple factors including maternal diabetes. Here we aimed to identify maternal diabetes-associated changes in placentas and analyzed placental gene expression to understand its modulation by maternal diabetes and birth mode. METHODS: Placental RNAseq transcriptome analyses were performed on maternally-derived decidua and fetal-derived villous tissue from pregnancies of mothers with type 1 diabetes (n = 14), gestational diabetes (n = 6) and without diabetes (n = 14). Information on delivery mode and anesthesia were included as covariables. Analyses were performed separately for decidua and fetal tissues and adjusted for sex. RESULTS: Substantial placenta gene expression variation was associated with factors other than maternal diabetes, including site, sex, anesthesia type and delivery mode. Two dominant gene expression clusters aligned to anesthesia and delivery mode were observed for decidua and villous tissue. Upregulation of genes within pathways related to organ morphogenesis and downregulation of immune response to steroid- and hypoxia pathway genes was characteristic of placentas from primary cesarean section deliveries with spinal anesthesia. Opposite profiles were observed for placentas from secondary cesarean and epidural anesthesia deliveries. Placentas from vaginal delivery had intermediate gene expression profiles. More subtle changes were associated with maternal diabetes: upregulation of ribosome activity, down-regulation of maternally-derived decidua chemokine signaling pathways and for gestational diabetes, alteration in hypoxia response genes. DISCUSSION: The findings reveal suppression of immune pathways and upregulation of ribosome activity in the placenta by maternal diabetes highlighting the importance of confounding factors when examining cell and tissue expression profiles. Further studies should determine whether the observed gene expression differences are related to underlying causes for cesarean section deliveries.

Breast milk fatty acid profile in relation to infant growth and body composition: results from the INFAT study.

BACKGROUND: There is some evidence that the n-6/n-3 long-chain polyunsaturated fatty acids (LCPUFAs) ratio in early nutrition, and thus in breast milk, could influence infant body composition. METHODS: In an open-label randomized controlled trial (RCT), 208 healthy pregnant women were allocated to a dietary intervention (supplementation with 1,200 mg n-3 LCPUFAs per day and instructions to reduce arachidonic acid (AA) intake) from the 15th wk of gestation until 4 mo of lactation or to follow their habitual diet. Breast milk LCPUFAs at 6 wk and 4 mo postpartum were related to infant body composition assessed by skinfold thickness (SFT) measurements and ultrasonography during the first year of life. RESULTS: Dietary intervention significantly reduced breast milk n-6/n-3 LCPUFAs ratio. In the whole sample, early breast milk docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and n-3 LCPUFAs at 6 wk postpartum were positively related to the sum of four SFT measurements at age 1. Breast milk AA and n-6 LCPUFAs at 6 wk postpartum were negatively associated with weight, BMI, and lean body mass (LBM) up to 4 mo postpartum. CONCLUSION: Breast milk n-3 LCPUFAs appear to stimulate fat mass growth over the first year of life, whereas AA seems to be involved in the regulation of overall growth, especially in the early postpartum period.

Fetal sex modulates placental microRNA expression, potential microRNA-mRNA interactions, and levels of amino acid transporter expression and substrates: INFAT study subpopulation analysis of n-3 LCPUFA intervention during pregnancy and associations with offspring body composition.

BACKGROUND: Previously, we revealed sexually dimorphic mRNA expression and responsiveness to maternal dietary supplementation with n-3 long-chain polyunsaturated fatty acids (LCPUFA) in placentas from a defined INFAT study subpopulation. Here, we extended these analyses and explored the respective placental microRNA expression, putative microRNA-mRNA interactions, and downstream target processes as well as their associations with INFAT offspring body composition. RESULTS: We performed explorative placental microRNA profiling, predicted microRNA-mRNA interactions by bioinformatics, validated placental target microRNAs and their putative targets by RT-qPCR and western blotting, and measured amino acid levels in maternal and offspring cord blood plasma and placenta. microRNA, mRNA, protein, and amino acid levels were associated with each other and with offspring body composition from birth to 5 years of age. Forty-six differentially regulated microRNAs were found. Validations identified differential expression for microRNA-99a (miR-99a) and its predicted target genes mTOR, SLC7A5, encoding L-type amino acid transporter 1 (LAT1), and SLC6A6, encoding taurine transporter (TauT), and their prevailing significant sexually dimorphic regulation. Target mRNA levels were mostly higher in placentas from control male than from female offspring, whereas respective n-3 LCPUFA responsive target upregulation was predominantly found in female placentas, explaining the rather balanced expression levels between the sexes present only in the intervention group. LAT1 and TauT substrates tryptophan and taurine, respectively, were significantly altered in both maternal plasma at 32 weeks' gestation and cord plasma following intervention, but not in the placenta. Several significant associations were observed for miR-99a, mTOR mRNA, SLC7A5 mRNA, and taurine and tryptophan in maternal and cord plasma with offspring body composition at birth, 1 year, 3 and 5 years of age. CONCLUSIONS: Our data suggest that the analyzed targets may be part of a sexually dimorphic molecular regulatory network in the placenta, possibly modulating gene expression per se and/or counteracting n-3 LCPUFA responsive changes, and thereby stabilizing respective placental and fetal amino acid levels. Our data propose placental miR-99, SLC7A5 mRNA, and taurine and tryptophan levels in maternal and fetal plasma as potentially predictive biomarkers for offspring body composition.

Maternal Type 1 Diabetes Reduces Autoantigen-Responsive CD4+ T Cells in Offspring.

Autoimmunity against pancreatic ß-cell autoantigens is a characteristic of childhood type 1 diabetes (T1D). Autoimmunity usually appears in genetically susceptible children with the development of autoantibodies against (pro)insulin in early childhood. The offspring of mothers with T1D are protected from this process. The aim of this study was to determine whether the protection conferred by maternal T1D is associated with improved neonatal tolerance against (pro)insulin. Consistent with improved neonatal tolerance, the offspring of mothers with T1D had reduced cord blood CD4+ T-cell responses to proinsulin and insulin, a reduction in the inflammatory profile of their proinsulin-responsive CD4+ T cells, and improved regulation of CD4+ T cell responses to proinsulin at 9 months of age, as compared with offspring with a father or sibling with T1D. Maternal T1D was also associated with a modest reduction in CpG methylation of the INS gene in cord blood mononuclear cells from offspring with a susceptible INS genotype. Our findings support the concept that a maternal T1D environment improves neonatal immune tolerance against the autoantigen (pro)insulin.

Gene variants of monocyte chemoattractant protein 1 and components of metabolic syndrome in KORA S4, Augsburg.

OBJECTIVE: Monocyte chemoattractant protein 1 (MCP-1) has been suggested to be involved in the development of several components of metabolic syndrome (MetS). The present study investigated the association of nine MCP-1 single nucleotide polymorphisms (SNPs) with MetS, type 2 diabetes mellitus and metabolic risk factors. SUBJECTS AND METHODS: The population-based study sample comprised 1630 subjects aged 55-74 years from KORA S4 (Cooperative Health Research in the Region of Augsburg Survey 4). Genotyping was carried out by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis of allele-dependent primer extension products. RESULTS: The MCP-1 SNP c.-3813C>T exhibited trends for differences between the genotype groups in triglycerides, 2-h glucose and uric acid (P = 0.0084, 0.014, 0.027). Other trends were observed for c.-928G>C associated with height and fasting glucose (P = 0.0024, 0.033), for c.105T>C with height and leukocytes (P = 0.0095, 0.047), for c.*65C>T and c.*3879C>T with MCP-1 levels (both P = 0.012) and for c.-2138A>T with interleukin-6 levels. After correction for multiple testing, none of the analysed SNPs, except c.-928G>C in men showed a significant association with MetS, T2DM or other analysed parameters. Haplotype MCP-1*1 and c.-928G>C in men (P = 0.0002, 0.0004) were significantly associated with an increase in height. CONCLUSIONS: This is the first study to investigate the associations of MCP-1 SNPs with MetS. We found trends for several components of MetS. These parameters were hyperlipidaemia, fasting and 2-h glucose, and uric acid. A new finding is that MCP-1*1 haplotype is associated with height. Further investigation in larger populations is needed to clarify the involvement of MCP-1 in MetS.

Type 1 diabetes vaccine candidates promote human Foxp3(+)Treg induction in humanized mice.

Immune tolerance is executed partly by Foxp3(+)regulatory T (Treg) cells, which suppress autoreactive T cells. In autoimmune type 1 diabetes (T1D) impaired tolerance promotes destruction of insulin-producing ß-cells. The development of autoantigen-specific vaccination strategies for Foxp3(+)Treg-induction and prevention of islet autoimmunity in patients is still in its infancy. Here, using human haematopoietic stem cell-engrafted NSG-HLA-DQ8 transgenic mice, we provide direct evidence for human autoantigen-specific Foxp3(+)Treg-induction in vivo. We identify HLA-DQ8-restricted insulin-specific CD4(+)T cells and demonstrate efficient human insulin-specific Foxp3(+)Treg-induction upon subimmunogenic vaccination with strong agonistic insulin mimetopes in vivo. Induced human Tregs are stable, show increased expression of Treg signature genes such as Foxp3, CTLA4, IL-2Rα and TIGIT and can efficiently suppress effector T cells. Such Foxp3(+)Treg-induction does not trigger any effector T cells. These T1D vaccine candidates could therefore represent an expedient improvement in the challenge to induce human Foxp3(+)Tregs and to develop novel precision medicines for prevention of islet autoimmunity in children at risk of T1D.

Effect of reducing the n-6/n-3 fatty acid ratio on the maternal and fetal leptin axis in relation to infant body composition.

OBJECTIVE: To investigate the effect of reducing the n-6/n-3 fatty acid ratio in maternal nutrition on the maternal and cord blood leptin axis and their association with infant body composition up to 2 years. DESIGN AND METHODS: 208 healthy pregnant women were randomized to either a dietary intervention to reduce the n-6/n-3 fatty acid ratio from 15th week of gestation until 4 months postpartum or a control group. Leptin, soluble leptin receptor and free leptin index were determined in maternal and cord plasma and related to infant body composition assessed by skinfold thicknesses up to 2 years. RESULTS: The intervention had no effect on either the maternal or fetal leptin axis. Maternal leptin in late pregnancy was inversely related to infant weight and lean body mass (LBM) up to 2 years, after multiple adjustments. Cord leptin was positively related to weight, body fat, and LBM at birth, and inversely associated with weight, BMI, fat mass, and LBM at 2 years and weight gain up to 2 years. The contribution of cord leptin to infant outcomes was overall stronger compared with maternal leptin. CONCLUSIONS: Both, maternal and fetal leptin were associated with subsequent infant anthropometry with a greater impact of fetal leptin.

Effect of reducing the n-6:n-3 long-chain PUFA ratio during pregnancy and lactation on infant adipose tissue growth within the first year of life: an open-label randomized controlled trial.

BACKGROUND: The composition of long-chain PUFAs (LCPUFAs) in the maternal diet may affect obesity risk in the mother's offspring. OBJECTIVE: We hypothesized that a reduction in the n-6 (omega-6):n-3 (omega-3) LCPUFA ratio in the diet of pregnant women and breastfeeding mothers may prevent expansive adipose tissue growth in their infants during the first year of life. DESIGN: In a randomized controlled trial, 208 healthy pregnant women were randomly assigned to an intervention (1200 mg n-3 LCPUFAs as a supplement per day and a concomitant reduction in arachidonic acid intake) or a control diet from the 15th wk of pregnancy to 4 mo of lactation. The primary outcome was infant fat mass estimated by skinfold thickness (SFT) measurements at 4 body sites at 3-5 d, 6 wk, and 4 and 12 mo postpartum. Secondary endpoints included sonographic assessment of abdominal subcutaneous and preperitoneal fat, fat distribution, and child growth. RESULTS: Infants did not differ in the sum of their 4 SFTs at ≤1 y of life [intervention: 24.1 ± 4.4 mm (n = 85); control: 24.1 ± 4.1 mm (n = 80); mean difference: -0.0 mm (95% CI: -1.3, 1.3 mm)] or in growth. Likewise, longitudinal ultrasonography showed no significant differences in abdominal fat mass or fat distribution. CONCLUSIONS: We showed no evidence that supplementation with n-3 fatty acids and instructions to reduce arachidonic acid intake during pregnancy and lactation relevantly affects fat mass in offspring during the first year of life. Prospective long-term studies are needed to explore the efficacy of this dietary approach for primary prevention. This trial was registered at clinicaltrials.gov as NCT00362089.

Functional characterization of promoter variants of the adiponectin gene complemented by epidemiological data.

OBJECTIVE: Adiponectin (APM1, ACDC) is an adipocyte-derived protein with downregulated expression in obesity and insulin-resistant states. Several potentially regulatory single nucleotide polymorphisms (SNPs) within the APM1 gene promoter region have been associated with circulating adiponectin levels. None of them have been functionally characterized in adiponectin-expressing cells. Hence, we investigated three SNPs (rs16861194, rs17300539, and rs266729) for their influence on adiponectin promoter activity and their association with circulating adiponectin levels. RESEARCH DESIGN AND METHODS: Basal and rosiglitazone-induced promoter activity of different SNP combinations (haplotypes) was analyzed in 3T3-L1 adipocytes using luciferase reporter gene assays and DNA binding studies comparing all possible APM1 haplotypes. This functional approach was complemented with analysis of epidemiological population-based data of 1,692 participants of the MONICA/KORA S123 cohort and 696 participants from the KORA S4 cohort for SNP and haplotype association with circulating adiponectin levels. RESULTS: Major to minor allele replacements of the three SNPs revealed significant effects on promoter activity in luciferase assays. Particularly, a minor variant in rs16861194 resulted in reduced basal and rosiglitazone-induced promoter activity and hypoadiponectinemia in the epidemiological datasets. The haplotype with the minor allele in all three SNPs showed a complete loss of promoter activity, and no subject carried this haplotype in either of the epidemiological samples (combined P value for statistically significant difference from a random sample was 0.006). CONCLUSIONS: Our results clearly demonstrate that promoter variants associated with hypoadiponectinemia in humans substantially affect adiponectin promoter activity in adipocytes. Our combination of functional experiments with epidemiological data overcomes the drawback of each approach alone.

APOA5 variants and metabolic syndrome in Caucasians.

Apolipoprotein A5 (APOA5) gene variants were reported to be associated with two components of metabolic syndrome (MetS): higher TG levels and lower HDL levels. Moreover, a recent Japanese case-control study found variant -1131T>C associated with MetS itself. Thus, our study systematically analyzed the APOA5 gene for association with lipid parameters, any other features of MetS, including waist circumference, glucose-related parameters, blood pressure, uric acid, and MetS itself in Caucasians. Ten polymorphisms were analyzed in a large fasting sample of the population-based Cooperative Health Research in the Region of Augsburg (KORA) survey S4 (n = 1,354; southern Germany) and in a second fasting sample, the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) study (n = 1,770; Austria). Minor alleles of variants -1131T>C, -3A>G, c.56C>G, 476G>A, and 1259T>C were significantly associated with higher TG levels in single polymorphism (P < 0.001) and haplotype (P G was associated with higher risk for MetS [odds ratio (95% confidence interval) = 1.43 (1.04, 1.99), P = 0.03 for KORA and 1.48 (1.10, 1.99), P = 0.009 for SAPHIR). Our study confirms the association of the APOA5 locus with TG and HDL levels in humans. Furthermore, the data suggest a different mechanism of APOA5 impact on MetS in Caucasians, as variant c.56C>G (not analyzed in the Japanese study) and not -1131T>C, as in the Japanese subjects, was associated with MetS.