Longitudinal,genome-scale analysis of DNA methylation in twins from birth to 18 months of age reveals rapid epigenetic change in early life and pair-specific effects of discordance

Background

The extent to which development- and age-associated epigenetic changes are influenced by genetic, environmental and stochastic factors remains to be discovered. Twins provide an ideal model with which to investigate these influences but previous cross-sectional twin studies provide contradictory evidence of within-pair epigenetic drift over time. Longitudinal twin studies can potentially address this discrepancy.

Results

In a pilot, genome-scale study of DNA from buccal epithelium, a relatively homogeneous tissue, we show that one-third of the CpGs assayed show dynamic methylation between birth and 18 months. Although all classes of annotated genomic regions assessed show an increase in DNA methylation over time, probes located in intragenic regions, enhancers and low-density CpG promoters are significantly over-represented, while CpG islands and high-CpG density promoters are depleted among the most dynamic probes. Comparison of co-twins demonstrated that within-pair drift in DNA methylation in our cohort is specific to a subset of pairs, who show more differences at 18 months. The rest of the pairs show either minimal change in methylation discordance, or more similar, converging methylation profiles at 18 months. As with age-associated regions, sites that change in their level of within-pair discordance between birth and 18 months are enriched in genes involved in development, but the average magnitude of change is smaller than for longitudinal change.

Conclusions

Our findings suggest that DNA methylation in buccal epithelium is influenced by non-shared stochastic and environmental factors that could reflect a degree of epigenetic plasticity within an otherwise constrained developmental program.     

Arterial stiffness in hypertensive and type 2 diabetes patients in Ghana: comparison of the cardio-ankle vascular index and central aortic techniques

Background

Diabetes and hypertension increase arterial stiffness and cardiovascular events in all societies studied so far; sub-Saharan African studies are sparse. We investigated factors affecting arterial function in Ghanaians with diabetes, hypertension, both or neither.

Method

Testing the hypothesis that arterial stiffness would progressively increase from controls to multiply affected patients, 270 participants were stratified into those with diabetes or hypertension only, with both, or without either. Cardio-ankle vascular index (CAVI), heart–ankle pulse wave velocity (haPWV), aortic PWV (PWVao) by Arteriograph, aortic and brachial blood pressures (BP), were measured.

Results

In patients with both diabetes and hypertension compared with either alone, values were higher of CAVI (mean?±?SD, 8.3?±?1.2 vs 7.5?±?1.1 and 7.4?±?1.1 units; p?<?0.05), PWVao (9.1?±?1.4 vs 8.7?±?1.9 and 8.1?±?0.9 m/s; p?<?0.05) and haPWV (8.5?±?1 vs 7.9?±?1 and 7.2?±?0.7 m/s; p?<?0.05) respectively. In multivariate analysis, age, having diabetes or hypertension and BMI were independently associated with CAVI in all participants (β?=?0.49, 0.2, 0.17 and -0.2 units; p?<?0.01, respectively). Independent determinants of PWVao were heart rate, systolic BP and age (β?=?0.42, 0.27 and 0.22; p?<?0.01), and for haPWV were systolic BP, age, BMI, diabetes and hypertension status (β?=?0.46, 0.32, -0.2, 0.2 and 0.11; p?<?0.01).

Conclusion

In this sub-Saharan setting with lesser atherosclerosis than the western world, arterial stiffness is significantly greater in patients with coexistent diabetes and hypertension but did not differ between those with either diabetes or hypertension only. Simple, reproducibly measured PWV/CAVI may offer effective and efficient targets for intervention.

     

Peanut Stripe Potyvirus Resistance in Peanut (Arachis Hypogaea L.) Plants Carrying Viral Coat Protein Gene Sequences

Peanut (Arachis hypogaea L.) lines exhibiting high levels of resistance to peanut stripe virus (PStV) were obtained following microprojectile bombardment of embryogenic callus derived from mature seeds. Fertile plants of the commercial cultivars Gajah and NC7 were regenerated following co-bombardment with the hygromycin resistance gene and one of two forms of the PStV coat protein (CP) gene, an untranslatable, full length sequence (CP2) or a translatable gene encoding a CP with an N-terminal truncation (CP4). High level resistance to PStV was observed for both transgenes when plants were challenged with the homologous virus isolate. The mechanism of resistance appears to be RNA-mediated, since plants carrying either the untranslatable CP2 or CP4 had no detectable protein expression, but were resistant or immune (no virus replication). Furthermore, highly resistant, but not susceptible CP2 T0 plants contained transgene-specific small RNAs. These plants now provide important germplasm for peanut breeding, particularly in countries where PStV is endemic and poses a major constraint to peanut production.     

Two domains of the epstein-barr virus origin DNA-binding protein, EBNA1, orchestrate sequence-specific DNA binding

The EBNA1 (for Epstein-Barr nuclear antigen 1) protein of Epstein-Barr virus governs the replication and partitioning of the viral genomes during latent infection by binding to specific recognition sites in the viral origin of DNA replication. The crystal structure of the DNA binding portion of the EBNA1 protein revealed that this region comprises two structural motifs; a core domain, which mediates protein dimerization and is structurally homologous to the DNA binding domain of the papillomavirus E2 protein, and a flanking domain, which mediated all the observed sequence-specific contacts. To test the possibility that the EBNA1 core domain plays a role in sequence-specific DNA binding not revealed in the crystal structure, we examined the effects of point mutations in potential hydrogen bond donors located in an alpha-helix of the EBNA1 core domain whose structural homologue in E2 mediates sequence-specific DNA binding. We show that these mutations severely reduce the affinity of EBNA1 for its recognition site, and that the core domain, when expressed in the absence of the flanking domain, has sequence-specific DNA binding activity. Flanking domain residues were also found to contribute to the DNA binding activity of EBNA1. Thus, both the core and flanking domains of EBNA1 play direct roles in DNA recognition.     

Altered response of a human squamous cell carcinoma cell line to 1, 25-dihydroxyvitamin D(3) after transfer of a normal chromosome 11

Previous work in our laboratory using functional assays for tumorigenicity identified a tumor suppressor element on human chromosome 11q for the cutaneous squamous cell carcinoma cell line A388.6TG.c2. In this report, we screened a variety of agents for differential effects on A388.6TG.c2 compared to a growth-suppressed chromosome 11 microcell hybrid of A388.6TG.c2. One of the agents, 1, 25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3); calcitriol), exerted a growth-altering effect on A388.6TG.c2, which formed rounded cell clusters across the surface of the raft by Day 6 of treatment. In contrast, full-length chromosome 11 hybrids of A388.6TG.c2, as well as two other squamous cell carcinoma cell lines (FaDu and A431), when treated with 1,25(OH)(2)D(3), failed to demonstrate this cell-clumping phenotype. To pursue the hypothesis that the growth suppressor element is involved in altering the response to 1, 25(OH)(2)D(3), we tested microcell hybrids carrying t(X;11) chromosomes lacking large portions of 11q. Although these hybrids, like the parent A388.6TG.c2 cells, demonstrated extensive growth in organotypic cultures, they failed to form cell clusters with 1, 25(OH)(2)D(3) treatment. These results suggest that the chromosome 11 element that alters the response to 1,25(OH)(2)D(3) is distinct from the growth-suppressing element. An examination of differentiation marker expression revealed identical patterns of basal and suprabasal markers for A388.6TG.c2 and for a chromosome 11 hybrid with or without treatment with 1,25(OH)(2)D(3). Finally, characterization of candidate tumor suppressor gene PPP2R1B, which encodes for a subunit of protein phosphatase 2A (PP2A), showed seemingly insignificant alterations by cDNA sequence analysis. Collectively, the data suggest that human chromosome 11 contains two different tumor suppressor elements that may account for the two areas of loss of heterozygosity observed on the long arm of this chromosome.