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

Published: May 6th, 2022

DOI:

Natália Yumi Noronha^1*, Guilherme da Silva Rodrigues^1*, Marcela Augusta de Souza Pinhel¹, Jean-Baptiste Cazier^2,3, Lígia Moriguchi Watanabe¹, Albert Nobre Menezes⁴, Carlos Roberto Bueno⁵, Carolina Ferreira Nicoletti¹, Bruno Affonso Parenti de Oliveira¹, Isabelle Mello Schineider⁶, Isabella Harumi Yonehara Noma⁷, Igor Caetano Dias Alcarás⁸, Fernando Barbosa⁹, Carla Barbosa Nonino⁶

¹Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, ²Centre for Computational Biology, University of Birmingham, ³Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, ⁴Cancer Genetics and Evolution Laboratory, Cancer Research UK, Institute of Genetics & Molecular Medicine, The University of Edinburgh, ⁵Ribeirão Preto School of Physical Education and Sport, University of São Paulo, ⁶Health Sciences Department, Ribeirao Preto Medical School, University of Sao Paulo, ⁷Faculty of Pharmaceutical Sciences, University of São Paulo, ⁸Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, ⁹Department of Clinical, Bromatological and Toxicological Analysis, Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of São Paulo

* These authors contributed equally

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.