Sample Preparation to Bioinformatics Analysis of DNA Methylation: Association Strategy for Obesity and Related Trait Studies.

Obesity is directly connected to lifestyle and has been associated with DNA methylation changes that may cause alterations in the adipogenesis and lipid storage processes contributing to the development of the disease. We demonstrate a complete protocol from selection to epigenetic data analysis of patients with and without obesity. All steps from the protocol were tested and validated in a pilot study. 32 women participated in the study, in which 15 individuals were classified with obesity according to Body Mass Index (BMI) (45.1 ± 5.4 kg/m2); and 17 individuals were classified without obesity according to BMI (22.6 ± 1.8 kg/m2). In the group with obesity, 564 CpG sites related to fat mass were identified by linear regression analysis. The CpG sites were in the promoter regions. The differential analysis found 470 CpGs hypomethylated and 94 hypermethylated sites in individuals with obesity. The most hypomethylated enriched pathwayswere in the RUNX, WNT signaling, and response to hypoxia. The hypermethylated pathways were related to insulin secretion, glucagon signaling, and Ca2+. We conclude that the protocol effectively identified DNA methylation patterns and trait-related DNA methylation. These patterns could be associated with altered gene expression, affecting adipogenesis and lipid storage. Our results confirmed that an obesogenic lifestyle could promote epigenetic changes in human DNA.

Mutations, Differential Gene Expression, and Chimeric Transcripts in Esophageal Squamous Cell Carcinoma Show High Heterogeneity.

Esophageal squamous cell carcinoma (ESCC) is a frequent and lethal neoplasia. As recent advances in targeted therapy have not improved ESCC prognosis, characterization of molecular alterations associated to this tumor is of foremost relevance. In this study, we analyze, for the first time, the complete genomic profile of ESCC by RNA-seq. TP53 was the most frequently mutated gene in the investigation and validation sets (78.6% and 67.4%, respectively). Differential expression analysis between tumor and nontumor adjacent mucosa showed 6698 differentially expressed genes, most of which were overexpressed (74%). Enrichment analysis identified overrepresentation of Wnt pathway, with overexpressed activators and underexpressed inactivators, suggesting activation of canonical and noncanonical Wnt signaling pathways. Higher WNT7B expression was associated with poor prognosis. Twenty-one gene fusions were identified in 50% of tumors, none of which involving the same genes in different patients; 71% of fusions involved syntenic genes. Comparisons with TCGA data showed co-amplification of seven gene pairs involved in fusions in the present study (~33%), suggesting that these rearrangements might have been driven by chromoanagenesis. In conclusion, genomic alterations in ESCC are highly heterogeneous, impacting negatively in target therapy development.

MECP2, a gene associated with Rett syndrome in humans, shows conserved coding regions, independent Alu insertions, and a novel transcript across primate evolution.

BACKGROUND: The methyl-CpG Binding Protein two gene (MECP2) encodes a multifunctional protein comprising two isoforms involved in nuclear organization and regulation of splicing and mRNA template activity. This gene is normally expressed in all tissues, with a higher expression level in the brain during neuronal maturation. Loss of MECP2 function is the primary cause of Rett syndrome (RTT) in humans, a dominant, X-linked disorder dramatically affecting neural and motor development. RESULTS: We investigated the molecular evolution of MECP2 in several primate taxa including 36 species in 16 genera of neotropical (platyrrhine) primates. The coding region of the MECP2_e2 isoform showed a high level of evolutionary conservation among humans and other primates, with amino acid substitutions in 14 codons and one in-frame insertion of a single serine codon, between codons 357 and 358, in Ateles paniscus. Most substitutions occurred in noncritical regions of MECP2 and the majority of the algorithms used for analyzing selection did not provide evidence of positive selection. Conversely, we found 48 sites under negative selection in different regions, 23 of which were consistently found by three different algorithms. Similar to an inverted Alu insert found previously in a lesser ape at a parallel location, one Alu insertion of approximately 300 bp in Cebus and Sapajus was found in intron 3. Phylogenetic reconstruction of the intron 3 data provided a topology that was coincident with the consensus arrangement of the primate taxa. RNAseq data in the neotropical primate Callimico goeldii revealed a novel transcript consisting of a noncontinuous region of the human-homologous intron 2 in this species; this transcript accounted for two putative polypeptides. CONCLUSIONS: Despite the remarkable evolutionary conservation of MECP2, one in-frame codon insertion was observed in A. paniscus, and one region of intron 3 was affected by a trans-specific Alu retrotransposition in two neotropical primate genera. Moreover, identification of novel MECP2 transcripts in Callimico suggests that part of a homologous human intronic region might be expressed, and that the potential open reading frame in this region might be a subject of interest in RTT patients who carry an apparently normal MECP2 sequence.