Differential SLC6A4 methylation: a predictive epigenetic marker of adiposity from birth to adulthood.

BACKGROUND: The early life environment may influence susceptibility to obesity and metabolic disease in later life through epigenetic processes. SLC6A4 is an important mediator of serotonin bioavailability, and has a key role in energy balance. We tested the hypothesis that methylation of the SLC6A4 gene predicts adiposity across the life course. METHODS: DNA methylation at 5 CpGs within the SLC6A4 gene identified from a previous methyl binding domain array was measured by pyrosequencing. We measured DNA methylation in umbilical cord (UC) from children in the Southampton Women's Survey cohort (n = 680), in peripheral blood from adolescents in the Western Australian Pregnancy Cohort Study (n = 812), and in adipose tissue from lean and obese adults from the UK BIOCLAIMS cohort (n = 81). Real-time PCR was performed to assess whether there were corresponding alterations in gene expression in the adipose tissue. RESULTS: Lower UC methylation of CpG5 was associated with higher total fat mass at 4 years (p = 0.031), total fat mass at 6-7 years (p = 0.0001) and % fat mass at 6-7 years (p = 0.004). Lower UC methylation of CpG5 was also associated with higher triceps skinfold thickness at birth (p = 0.013), 6 months (p = 0.038), 12 months (p = 0.062), 2 years (p = 0.0003), 3 years (p = 0.00004) and 6-7 years (p = 0.013). Higher maternal pregnancy weight gain (p = 0.046) and lower parity (p = 0.029) were both associated with lower SLC6A4 CpG5 methylation. In adolescents, lower methylation of CpG5 in peripheral blood was associated with greater concurrent measures of adiposity including BMI (p ≤ 0.001), waist circumference (p = 0.011), subcutaneous fat (p ≤ 0.001) and subscapular, abdominal and suprailiac skinfold thicknesses (p = 0.002, p = 0.008, p = 0.004, respectively). In adipose tissue, methylation of both SLC6A4 CpG5 (p = 0.019) and expression of SLC6A4 (p = 0.008) was lower in obese compared with lean adults. CONCLUSIONS: These data suggest that altered methylation of CpG loci within SLC6A4 may provide a robust marker of adiposity across the life course.

DNA methylation of intragenic CpG islands depends on their transcriptional activity during differentiation and disease.

The human genome contains ∼30,000 CpG islands (CGIs). While CGIs associated with promoters nearly always remain unmethylated, many of the ∼9,000 CGIs lying within gene bodies become methylated during development and differentiation. Both promoter and intragenic CGIs may also become abnormally methylated as a result of genome rearrangements and in malignancy. The epigenetic mechanisms by which some CGIs become methylated but others, in the same cell, remain unmethylated in these situations are poorly understood. Analyzing specific loci and using a genome-wide analysis, we show that transcription running across CGIs, associated with specific chromatin modifications, is required for DNA methyltransferase 3B (DNMT3B)-mediated DNA methylation of many naturally occurring intragenic CGIs. Importantly, we also show that a subgroup of intragenic CGIs is not sensitive to this process of transcription-mediated methylation and that this correlates with their individual intrinsic capacity to initiate transcription in vivo. We propose a general model of how transcription could act as a primary determinant of the patterns of CGI methylation in normal development and differentiation, and in human disease.

Association between perinatal methylation of the neuronal differentiation regulator HES1 and later childhood neurocognitive function and behaviour.

BACKGROUND: Early life environments induce long-term changes in neurocognitive development and behaviour. In animal models, early environmental cues affect neuropsychological phenotypes via epigenetic processes but, as yet, there is little direct evidence for such mechanisms in humans. METHOD: We examined the relation between DNA methylation at birth and child neuropsychological outcomes in two culturally diverse populations using a genome-wide methylation analysis and validation by pyrosequencing. RESULTS: Within the UK Southampton Women's Survey (SWS) we first identified 41 differentially methylated regions of interest (DMROI) at birth associated with child's full-scale IQ at age 4 years. Associations between HES1 DMROI methylation and later cognitive function were confirmed by pyrosequencing in 175 SWS children. Consistent with these findings, higher HES1 methylation was associated with higher executive memory function in a second independent group of 200 SWS 7-year-olds. Finally, we examined a pathway for this relationship within a Singaporean cohort (n = 108). Here, HES1 DMROI methylation predicted differences in early infant behaviour, known to be associated with academic success. In vitro, methylation of HES1 inhibited ETS transcription factor binding, suggesting a functional role of this site. CONCLUSIONS: Thus, our findings suggest that perinatal epigenetic processes mark later neurocognitive function and behaviour, providing support for a role of epigenetic processes in mediating the long-term consequences of early life environment on cognitive development.

Differential pathways to adult metabolic dysfunction following poor nutrition at two critical developmental periods in sheep.

Epidemiological and experimental studies suggest early nutrition has long-term effects on susceptibility to obesity, cardiovascular and metabolic diseases. Small and large animal models confirm the influence of different windows of sensitivity, from fetal to early postnatal life, on offspring phenotype. We showed previously that undernutrition in sheep either during the first month of gestation or immediately after weaning induces differential, sex-specific changes in adult metabolic and cardiovascular systems. The current study aims to determine metabolic and molecular changes that underlie differences in lipid and glucose metabolism induced by undernutrition during specific developmental periods in male and female sheep. Ewes received 100% (C) or 50% nutritional requirements (U) from 1-31 days gestation, and 100% thereafter. From weaning (12 weeks) to 25 weeks, offspring were then fed either ad libitum (CC, UC) or were undernourished (CU, UU) to reduce body weight to 85% of their individual target. From 25 weeks, all offspring were fed ad libitum. A cohort of late gestation fetuses were studied after receiving either 40% nutritional requirements (1-31 days gestation) or 50% nutritional requirements (104-127 days gestation). Post-weaning undernutrition increased in vivo insulin sensitivity, insulin receptor and glucose transporter 4 expression in muscle, and lowered hepatic methylation at the delta-like homolog 1/maternally expressed gene 3 imprinted cluster in adult females, but not males. Early gestational undernutrition induced lower hepatic expression of gluconeogenic factors in fetuses and reduced in vivo adipose tissue insulin sensitivity in adulthood. In males, undernutrition in early gestation increased adipose tissue lipid handling mechanisms (lipoprotein lipase, glucocorticoid receptor expression) and hepatic methylation within the imprinted control region of insulin-like growth factor 2 receptor in adulthood. Therefore, undernutrition during development induces changes in mechanisms of lipid and glucose metabolism which differ between tissues and sexes dependent on the period of nutritional restriction. Such changes may increase later life obesity and dyslipidaemia risk.

Variation in telangiectasia predisposing genes is associated with overall radiation toxicity.

PURPOSE: In patients receiving radiotherapy for breast cancer where the heart is within the radiation field, cutaneous telangiectasiae could be a marker of potential radiation-induced heart disease. We hypothesized that single nucleotide polymorphisms (SNPs) in genes known to cause heritable telangiectasia-associated disorders could predispose to such late, normal tissue vascular damage. METHODS AND MATERIALS: The relationship between cutaneous telangiectasia as a late normal tissue radiation injury phenotype in 633 breast cancer patients treated with radiotherapy was examined. Patients were clinically assessed for the presence of cutaneous telangiectasia and genotyped at nine SNPs in three candidate genes. Candidate SNPs were within the endoglin (ENG) and activin A receptor, type II-like 1 (ACVRL1) genes, mutations in which cause hereditary hemorrhagic telangiectasia and the ataxia-telangiectasia mutated (ATM) gene associated with ataxia-telangiectasia. RESULTS: A total of 121 (19.1%) patients exhibited a degree of cutaneous telangiectasiae on clinical examination. Regression was used to examine the associations between the presence of telangiectasiae in patients who underwent breast-conserving surgery, controlling for the effects of boost and known brassiere size (n=388), and individual geno- or haplotypes. Inheritance of ACVRL1 SNPs marginally contributed to the risk of cutaneous telangiectasiae. Haplotypic analysis revealed a stronger association between inheritance of a ATM haplotype and the presence of cutaneous telangiectasiae, fibrosis and overall toxicity. No significant association was observed between telangiectasiae and the coinheritance of the candidate ENG SNPs. CONCLUSIONS: Genetic variation in the ATM gene influences reaction to radiotherapy through both vascular damage and increased fibrosis. The predisposing variation in the ATM gene will need to be better defined to optimize it as a predictive marker for assessing radiotherapy late effects.

Association between single nucleotide polymorphisms in the DNA repair gene LIG3 and acute adverse skin reactions following radiotherapy.

Many genes have been associated with radiotherapy toxicity, but most have only been found in a single study. Using our cohort of 480 breast cancer patients, we provide replicated evidence that a polymorphism near the LIG3 gene is associated with acute skin toxicity following radiotherapy.

Genetic basis of variation in tenofovir drug susceptibility in HIV-1.

OBJECTIVE: To develop an improved model for the genetic basis of reduced susceptibility to tenofovir in vitro. METHODS: A dataset of 532 HIV-1 subtype B reverse transcriptase genotypes for which matched phenotypic susceptibility data were available was assembled, both as a continuous (transformed) dataset and a categorical dataset generated by imposing a cut-off on the basis of earlier studies of in-vivo response of 1.4-fold. Models were generated using stepwise regression, decision tree and random forest approaches on both the continuous and categorical data. Models were compared by mean squared error (continuous models), or by misclassification rates by nested crossvalidation. RESULTS: From the continuous dataset, stepwise linear regression, regression tree and regression forest methods yielded models with MSE of 0.46, 0.48 and 0.42 respectively. Amino acids 215, 65, 41, 67, 184 and 151 in HIV-1 reverse transcriptase were identified in all three models and amino acid 210 in two. The categorical data yielded logistic regression, classification tree and forest models with misclassification rates of 26, 24 and 23%, respectively. Amino acids 215, 65 and 67 appeared in all; 41, 184, 210 and 151 were also included in the classification forest model. CONCLUSION: The random forests approach has yielded a substantial improvement in the available models to describe the genetic basis of reduced susceptibility to tenofovir in vitro. The most important sites in these models are amino acid sites 215, 65, 41, 67, 184, 151 and 210 in HIV-1 reverse transcriptase.

Trans-sodium crocetinate and hemorrhagic shock.

Trans-sodium crocetinate (TSC) has been found to alleviate the symptoms of hemorrhagic shock in that, after the drug is given to hemorrhaged rats, blood pressure rises, elevated lactate levels are reduced, cellular damage in the liver and kidney is less, and survival is increased. The mechanism of action proposed for TSC is that it increases the diffusion of oxygen through blood plasma and into the tissue. The study reported here explores another proposed mechanism, the scavenging of free radicals. It is shown that TSC does scavenge radicals and may be more efficient than other free-radical scavengers. However, this may not be the mechanism of action for TSC during hemorrhagic shock. Not only are the TSC concentrations needed for radical scavenging greater than those which provide beneficial effects in hemorrhagic shock, but it is also shown that another radical scavenger, trolox, has no effect on oxygen consumption during shock. Trans-sodium crocetinate clearly holds promise as a useful treatment of hemorrhagic shock. Pharmacokinetics data are presented for different modes of administration. Initial studies suggest that instillation of TSC into the trachea or intramuscular injection may provide useful alternative treatment routes.

Oral insulin delivery using P(MAA-g-EG) hydrogels: effects of network morphology on insulin delivery characteristics.

Hydrogels of poly(methacrylic acid-g-ethylene glycol) were prepared using different reaction water contents in order to vary the network mesh size, swelling behavior and insulin loading/release kinetics. Gels prepared with greater reaction solvent contents swelled to a greater degree and had a larger network mesh size. All of the hydrogels were able to incorporate insulin and protected it from release in acidic media. At higher pH (7.4), the release rates increased with reaction solvent content. Using a closed loop animal model, all of the insulin loaded formulations produced significant insulin absorption in the upper small intestine combined with hypoglycemic effects. In these studies, bioavailabilities ranged from 4.6% to 7.2% and were dependent on reaction solvent content.