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Standardization of PCR and sequencing methods for DNA methylation analysis of LDLR, PCSK9 and LDLRAP1

Grant number: 19/16967-4
Support Opportunities:Scholarships in Brazil - Scientific Initiation
Effective date (Start): August 01, 2019
Effective date (End): January 17, 2021
Field of knowledge:Health Sciences - Pharmacy
Principal Investigator:Mario Hiroyuki Hirata
Grantee:Daiana Vitor de Oliveira Silva
Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Associated research grant:16/12899-6 - Genomics, epigenomics and pharmacogenomics characterization of familial hypercholesterolemia in the Brazilian population, AP.TEM


Familial Hypercholesterolemia (FH) is an autosomal dominant disease that affects cholesterol metabolism and results in decreasing of LDL catabolism and increasing of its plasma concentration. FH phenotypes are consequence of variants in HF-related genes like Low-Density Lipoprotein Receptor (LDLR), APOliprotein B (APOB), and Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9). However, other genes such as Low Density Lipoprotein Receptor Adapter Protein 1 (LDLRAP1) have been associated with different phenotypes in Autosomal Recessive Hypercholesterolaemia (ARH). Despite the advances in sequencing technologies, variations in DNA sequence do not explain all the heritability of blood lipid traits (only about 60%). In recent years, the phenotypes not associated to genomic structural variations are been investigated and epigenetic mechanisms such as DNA methylation, which are involved in the control of gene transcription and translation, may help to explain these phenomenon. DNA methylation is a process by which methyl groups (CH3) are added to the DNA molecule at CpG sites. In the genome, CpG sites may occur isolated or at CpG islands, in particular at gene promoter regions. DNA methylation in promoter region results in gene silencing by blocking binding of the transcription factors to the gene regulatory regions. Thus, epigenetic studies play an important role to better understand the pathophysiological mechanisms of the cardiovascular diseases and to the development of new methods for their prevention, prognosis and therapy. The aim of this study is to standardize the PCR and sequencing methods for DNA methylation analysis of LDLR, PCSK9 and LDLRAP1. Genomic DNA will be isolated using the QIAmp DNA Blood Maxi Kit (Qiagen) and bisulfite-converted using EpiTect Fast DNA Bisulfite Conversion kit (Qiagen). The converted DNA at concentration e15ng/µl will be amplified by PCR using PyroMark PCR Kit reagents (Qiagen) and PyroMark CpG Assays (Qiagen). In the PCR, the following parameters will be evaluated: (1) Annealing temperatures and concentration of each PyroMark CpG Assay; (2) Converted DNA Input. The pyrosequencing will be performed using the PyroMark Q24 platform (Qiagen). In this step, the following parameters will be evaluated: (1) PCR product input and (2) sequencing primer concentration.

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