RESUMO
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.
Assuntos
Hiperlipoproteinemia Tipo II , Lipídeos , Lipoproteínas HDL , GenéticaRESUMO
OBJECTIVES: We investigated the relationship between non-syndromic cleft lip/palate (NSCLP) and polymorphisms in methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), methionine synthase reductase (MTRR), and RFC1, as well as the corresponding interactions with environmental factors. SUBJECTS AND METHODS: One hundred and forty NSCLP patients and their mothers, as well as 175 control individuals and their mothers, were recruited. Information regarding smoking and alcohol consumption was recorded. Blood samples were obtained in order to measure serum folate and cobalamin, as well as, plasma total homocysteine concentrations and to extract DNA. Polymorphisms in MTHFR(677C>T and 1298A>C), MTR(2756A>G), MTR(66A>G), and RFC1(80A>G) were analyzed by PCR-restriction fragment length polymorphism. RESULTS: Among the patients, 59.5% had cleft lip and palate, 22.0% had cleft palate, and 18.5% had cleft lip only. Maternal alcohol consumption and reduced folic acid concentrations in both children and mothers (P < 0.001 and P = 0.003, respectively) were risk factors for NSCLP. Patients and their mothers carrying the MTHFR 667T allele showed lower serum folate than CC (P = 0.011 and P = 0.030, respectively). Mothers who carried the MTHFR 1298C allele exhibited increased risk of having a child with NSCLP, after adjusting for alcohol consumption (OR: 1.75, 95% CI: 1.03-2.99, P = 0.038). CONCLUSIONS: Reduced folic acid levels, alcohol consumption, and the MTHFR 677T and 1298C alleles may have contributed to NSCLP development in this sample population from Rio Grande do Norte.
Assuntos
Consumo de Bebidas Alcoólicas , Encéfalo/anormalidades , Fenda Labial/genética , Fissura Palatina/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Efeitos Tardios da Exposição Pré-Natal/genética , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Feminino , Ferredoxina-NADP Redutase/genética , Ácido Fólico/sangue , Interação Gene-Ambiente , Homocisteína/sangue , Humanos , Masculino , Polimorfismo Genético , Gravidez , Proteína de Replicação C/genética , Adulto JovemRESUMO
OBJECTIVE: The aim of this study was to investigate the contribution of 6 polymorphic variants of the MSX1 gene in non-syndromic cleft lip and/or palate (NSCL/P). METHODS: Three hundred and fifty-eight individuals (158 NSCL/P cases and 200 controls) were genotyped by TaqMan allelic discrimination using predesigned SNP assays. Statistical analyses were conducted using the software spss 15.0 and the r statistical suite. Haplotype block structure and haplotype frequencies were determined using the Haploview. A P-value of 0.05 and confidence interval of 95% were used for all of statistical tests. RESULTS: The patients with non-syndromic cleft lip and/or palate were characterized by similar distribution of MSX1 genotypes and allele in comparison to subjects without oral clefts (P > 0.05). Two haplotype blocks were constructed with polymorphisms of MSX1 gene and haplotypes formed showed a similar frequency in patients with and without oral clefts. CONCLUSIONS: The present study provides no evidence that MSX1 polymorphisms (rs3775261, rs1042484, rs12532, rs6446693, rs4464513 and rs1907998) play a major role in NSCL/P.
