Functional analysis of ldlr genetic variants identified in hypercholesterolemic patients in brazil, USING CRISPR/CAS9

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.

SNPs from BCHE and DRD3 genes associated to cocaine abuse amongst violent individuals from Sao Paulo, Brazil.

Violence and drug abuse are highly destructive phenomena found world-wide, especially in Brazil. They seem to rise proportionally to one another and possibly related. Additionally, genetics may also play a role in drug abuse. This study has focused on identifying the use of cocaine within postmortem cases arriving at the Institute of Legal Medicine of Sao Paulo as well as the presence of certain single nucleotide polymorphisms (SNPs) to better understand one's susceptibility to abuse the drug. Both hair and blood samples have been extracted through a simple methanol overnight incubation or a rapid dilute-and-shoot method, respectively. The samples were then analyzed using an UPLC-ESI-MS/MS and genotyped through RT-PCR. Statistical analyses were performed via SPSS software. From 105 postmortem cases, 53% and 51% of the cases shown to be positive for cocaine in hair and blood, respectively. Genetic wise, a significant difference has been observed for SNP rs4263329 from the BCHE gene with higher frequencies of the genotypes A/G and G/G seen in cocaine users (OR=8.91; 95%CI=1.58-50.21; p=0.01). Likewise, also SNP rs6280 from the DRD3 gene presented a significant association, with both genotypes T/C and C/C being more frequent in users (OR=4.96; 95% CI=1.07-23.02; p=0.04). To conclude, a rather high proportion of cocaine has been found, which may suggest a connotation between the use of the drug and risky/violent behaviors. Additionally, significant associations were also found within two SNPs related to cocaine use, however, due to several inherent limitations, these must be confirmed.

Análise funcional in silico das variantes no gene da APOB em pacientes com hipercolesterolemia familial / In silico functional analysis of variants in the APOB gene in patients with familial hypercholesterolemia

INTRODUÇÃO: O diagnóstico molecular da hipercolesterolemia familial (HF) é atribuído principalmente as variantes nos genes LDLR, LDLRAP1, APOB e PCSK9. O objetivo deste estudo foi realizar análise in silico para investigar o impacto de variantes sem descrição na literatura no gene APOB observado em pacientes com HF. MÉTODOS: Foram selecionados 141 indivíduos com diagnóstico clínico de HF. As variantes no gene da APOB foram selecionadas após sequenciamento dos éxons de 61 genes utilizando a plataforma MiSeq (Illumina). Os dados foram analisados nos programas Real Time Analysis, MiSeq Reporter, BaseSpace Sequence Hub e VariantStudio. Para a análise in silico, as sequências molde das moléculas da apoB-100 e o LDLr foram selecionadas por modelagem comparativa considerando o maior grau de identidade. As sequências proteicas foram alinhadas e os modelos 3D foram construídos utilizando os programas SEAVIEW e MODELLER v9.21. O gráfico de Ramachandran do modelo de menor energia apresenta 0,5% de outliers e análise de regiões de desordem, como principal validação. Os resultados das conformações de ancoragem foram analisados no software PyMol 2.1. Os estudos de docking molecular foram realizados para identificar o melhor complexo de conformação usando o servidor web clusPRO. RESULTADOS: Após a análise molecular dos 141 pacientes foram identificadas 7 variantes missenses sem descrição na literatura no gene APOB (c.433C>T, c.2630C>T, c.2950G>A, c.5743G>A, c.7367C>A, c.9880T>C e c.10780T>C). Os estudos de docking das variantes demonstraram uma maior afinidade entre o LDLr e a apoB-100 (c.2630C> T; Pro877Leu) em comparação com a proteína não mutada. A troca dos resíduos permaneceu como propriedade físico-química, e comparando as distâncias de ligação das proteínas não-mutadas (5Å) e mutadas (3,5Å), sugere-se uma maior afinidade do complexo (LDLr-apoB-100) para a leucina, tal fato é afirmado pela análise da região de desordens da apoB-100, onde a posição 877 está em uma região desorganizada e flexível. Esta maior afinidade poderia levar a uma menor dissociação intracelular deste complexo, resultando em uma alta taxa de degradação do LDLr pelas enzimas lisossômicas, levando ao aumento da concentração plasmática de LDLc. Para as outras variantes não houve alterações significativas. CONCLUSÃO: Os resultados sugerem que estudos in silico baseados na ferramenta de docking molecular podem melhorar o conhecimento da contribuição genética no desenvolvimento da doença HF. Além disso, a variante APOB c.2630C> T deve ser avaliada in vitropara validação do mecanismo proposto. (AU)