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Functional analysis of ldlr genetic variants identified in hypercholesterolemic patients in brazil, USING CRISPR/CAS9
Mori, A. A; Los, B; Oliveira, V. F; Bortolin, R. H; Freitas, R. C. Costa de; Ferreira, G. M; Borges, J. B; Bastos, G. M; Barbosa, T. K. A. A; Marçal, E. D. S. R; Hernandez, C. D; Fajardo, C. M; Hirata, M. H; Hirata, R. D. C.
Affiliation
  • Mori, A. A; University of São Paulo. São Paulo. BR
  • Los, B; University of São Paulo. São Paulo. BR
  • Oliveira, V. F; University of São Paulo. São Paulo. BR
  • Bortolin, R. H; University of São Paulo. São Paulo. BR
  • Freitas, R. C. Costa de; University of São Paulo. São Paulo. BR
  • Ferreira, G. M; University of São Paulo. São Paulo. BR
  • Borges, J. B; Dante Pazzanese Institute of Cardiology. São Paulo. BR
  • Bastos, G. M; Dante Pazzanese Institute of Cardiology. São Paulo. BR
  • Barbosa, T. K. A. A; University of São Paulo. São Paulo. BR
  • Marçal, E. D. S. R; University of São Paulo. São Paulo. BR
  • Hernandez, C. D; University of São Paulo. São Paulo. BR
  • Fajardo, C. M; University of São Paulo. São Paulo. BR
  • Hirata, M. H; University of São Paulo. São Paulo. BR
  • Hirata, R. D. C; University of São Paulo. São Paulo. BR
Atherosclerosis ; 331(E127): e127-e127, Aug., 2021.
Article in English | Sec. Est. Saúde SP, CONASS, SESSP-IDPCPROD, Sec. Est. Saúde SP | ID: biblio-1348595
Responsible library: BR79.1
ABSTRACT
BACKGROUND AND

AIMS:

Introduction:

The familial hypercholesterolemia (FH) is one of the main causes of cardiovascular diseases, and it is mainly caused by genetic variants at the low-density lipoprotein receptor (LDLR). Although ultrasequencing technology has allowed the identification of several genetic variants, few of them was functional analyzed. The CRISPR/ Cas9 tool promotes precise genetic editing and allows the creation of experimental models, therefore contributing to the functional validation process.

Aim:

To use the CRISPR/Cas9 tool to perform in vitro functional analysis of LDLR variants identified in FH patients.

METHODS:

Two missense LDLR variants were selected within a group of variants identified in FH patients, based on in silico data, the affected protein domain and MAF. Three sgRNAs were designed for each of the variants c.551G>A and c.1118G>A, to analyze the accuracy of the sgRNAs. The sgRNAs were inserted on PX458 plasmid, cloned, purified in E. coli DH5a, and then co-transfected with the DNA template at HepG2 cells. The DNAs templates were designed to contain the selected variants.

RESULTS:

HepG2 cells co-transfected with PX458 constructs and DNA templates showed considerably transfection rate, being possible to visualize it at fluorescence microscopy. However, it was noted that single transfection of sgRNAs showed a higher transfection efficiency than cotransfection.

CONCLUSIONS:

We designed sgRNA for c.551G>A and c.1118G>A variants, being able to analyze the transfection efficiency. In further steps, we will select new sgRNAs for LDLR variants that have not been described yet, and functional analysis will be performed to determine the clinical relevance of these variants.
Subject(s)
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Collection: National databases / Brazil Database: CONASS / Sec. Est. Saúde SP / SESSP-IDPCPROD Main subject: Hyperlipoproteinemia Type II / Lipids / Lipoproteins, HDL Country/Region as subject: South America / Brazil Language: English Journal: Atherosclerosis Year: 2021 Document type: Article / Congress and conference Institution/Affiliation country: Dante Pazzanese Institute of Cardiology/BR / University of São Paulo/BR
Search on Google
Collection: National databases / Brazil Database: CONASS / Sec. Est. Saúde SP / SESSP-IDPCPROD Main subject: Hyperlipoproteinemia Type II / Lipids / Lipoproteins, HDL Country/Region as subject: South America / Brazil Language: English Journal: Atherosclerosis Year: 2021 Document type: Article / Congress and conference Institution/Affiliation country: Dante Pazzanese Institute of Cardiology/BR / University of São Paulo/BR
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