Identification of structural variants (CNVs) and epigenetic changes in patients with stroke

Author(s)

Amanda Donatti

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
• Defense date: 2016-01-29

Examining board members

Íscia Teresinha Lopes Cendes; Wagner Mauad Avelar; Rodrigo Bazan

• Advisor: Íscia Teresinha Lopes Cendes; Rodrigo Secolin

Abstract

Stroke is a heterogeneous and multifactorial condition, responsible for blockage in blood flow to the brain. Stroke can be classified in two main groups: ischemic and hemorrhagic. Many studies demonstrate the presence of genetic factors on stroke. Meta-analysis studies revealed 19 genes related to ischemic stroke. However, functional variants were not identified in 8 candidate genes (HDAC9, CDKN2B-AS1, PDE4D, PITX2, ZFHX3, ANGPT1, AGTRL1 and ALOX5AP).In addition, in vivo studies show that abnormal methylation pattern in DNA can influence stroke development or changes in proteins related to this phenotype, which leads to increased risk of stroke. Although some studies demonstrate influence of genetic variants in hemorrhagic stroke, studies with this subtype are less recurrent due to its low population frequency and higher mortality rate when compared to ischemic events. Copy number variations (CNVs) are common mutations which have been identified as significant causes of change in gene expression. Few studies have demonstrated a relation between genes and CNVs for ischemic and hemorrhagic stroke. The main objectives of this study are: to identify epigenetic factor on promoter region of candidate genes to ischemic stroke; and to identify copy number variations in patients with hemorrhagic stroke. We use DNA sample from patients with stroke provided from Joinville/SC. Through bioinformatics analysis, we identified regions potentially methylated (CpGs) on promoter regions of candidate genes from ischemic stroke. These candidate genes had their promoter region analyzed by methyl-specific PCR (MS-PCR). To analyze CNVs in patients with hemorrhagic stroke, we used microarray chips based on SNPs (Genome-Wide Human SNP Array 6.0; Affymetrix Inc.), whose results were analyzed by Genotyping Console® Software (Affymetrix Inc.). Our methylation studies did not resulted in any significant findings, since the CpGs islands in the promoter region of candidate genes were not amplified using the conditions initially tested in this study. In the CNVs studies, we analyzed 45 samples from patients and 41 samples from controls and identified 19 genes potentially related to stroke. We validated these results in a group of Italian patients , analyzing 18 patients with hemorrhagic stroke. Overall, we identified 2 genes which were present in both cohorts: MACROD2 and NSF. Gene MACROD2 does not have variants described in the normal general population genetic database, which makes it a potential candidate gene to influence hemorrhagic stroke. In conclusion, we have demonstrated for the first time that CNVs in certain candidate genes may influence the risk for hemorrhagic stroke. Our findings were confirmed in two independent samples of patients with hemorrhagic stroke (AU)

• FAPESP's process: 13/24932-0 - Identifying Copy Number Variations (CNVs) and Epigenetic Changes in Patients with Stroke
• Grantee: Amanda Donatti
• Support Opportunities: Scholarships in Brazil - Master