Browsing by Author "Pinto Pereira, Andreia Sofia"
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- Familial Hypercholesterolaemia: molecular and functional study of LDLR mutationsPublication . Pinto Pereira, Andreia SofiaCardiovascular disease (CVD) remains the most common cause of death globally. Dyslipidaemia is one of the most important risk factors that leads to CVD. It can be due to a monogenic condition or to polygenic/environmental causes as diabetes, obesity, tobacco use, excess of alcohol or reduced physical activity. The identification of the individuals at risk and the distinction of these two types of dyslipidaemia is important for a correct cardiovascular risk assessment, counselling, and treatment reducing, this way, cardiovascular mortality. Familial hypercholesterolaemia (FH) is an autosomal dominant disorder of cholesterol metabolism. Most commonly, FH results from inherited defects in the Low-Density Lipoprotein Receptor Gene (LDLR) leading to increased levels of circulating LDL cholesterol and lipid accumulation in arteries and tendons. Mutations in other genes as the apolipoprotein B gene (APOB) and proprotein convertase subtilisin/kexin type 9 gene (PCSK9), are also responsible for FH. The distribution pattern of apolipoprotein E gene (APOE) polymorphisms affects the affinity to lipoprotein receptors and, consequently, the clearance of dietary fat from the blood, also causing dyslipidaemia.
- Familial Hypercholesterolaemia: molecular and functional study of LDLR mutationsPublication . Pinto Pereira, Andreia Sofia; Bourbon, MafaldaCardiovascular disease (CVD) remains the most common cause of death globally. Dyslipidaemia is one of the most important risk factors that leads to CVD. It can be due to a monogenic condition or to polygenic/environmental causes as diabetes, obesity, tobacco use, excess of alcohol or reduced physical activity. The identification of the individuals at risk and the distinction of these two types of dyslipidaemia is important for a correct cardiovascular risk assessment, counselling, and treatment reducing, this way, cardiovascular mortality. Familial hypercholesterolaemia (FH) is an autosomal dominant disorder of cholesterol metabolism. Most commonly, FH results from inherited defects in the Low-Density Lipoprotein Receptor Gene (LDLR) leading to increased levels of circulating LDL cholesterol and lipid accumulation in arteries and tendons. Mutations in other genes as the apolipoprotein B gene (APOB) and proprotein convertase subtilisin/kexin type 9 gene (PCSK9), are also responsible for FH. The distribution pattern of apolipoprotein E gene (APOE) polymorphisms affects the affinity to lipoprotein receptors and, consequently, the clearance of dietary fat from the blood, also causing dyslipidaemia. The homozygous form of FH is rare and more severe, but the heterozygous form is common, with a frequency of 1/500 in most of European countries, although underdiagnosed in several populations, including the portuguese. FH is characterized by increased levels of plasmatic cholesterol since birth, which results in cholesterol deposits in extravascular tissues that can be identified in young patients (below 45 years old): xanthelasma, corneal arcus deposits and tendon xanthomas. This accumulation can cause premature arteriosclerosis and coronary heart disease (CHD). The presence of tendon xanthomas allows the differentiation of FH from other causes of hypercholesterolaemia as polygenic hypercholesterolaemia. More than 1700 different alterations in LDLR gene have been described worldwide. However, the functional studies for the great majority of these variants, have not been performed. For patients carrying these variants, a definitive molecular diagnosis for FH is not possible, representing a serious problem for FH diagnosis. In 1999, the Portuguese FH study was established at the National Institute of Health to identify the genetic cause of hypercholesterolaemia in individuals with a clinical diagnosis of FH. Index patients are included in this study using an adaptation of the Simon Broome (SB) criteria. Nonetheless, FH remains underdiagnosed and undertreated in the portuguese population. The main aim of this project was to perform the molecular identification of genetic variants in LDLR, APOB and APOE genes, causing dyslipidaemia in patients referred to the Portuguese FH Study in 2015/2016 with a clinical diagnosis of FH, in order to improve the identification of individuals at risk. Functional studies in RNA for putative splicing variants were also performed. The molecular diagnosis was performed for 60 index cases. Genomic DNA was isolated from peripheral blood lymphocytes using the salting out method. The 18 exons and promotor region of LDLR, part of exons 26 and 29 of APOB and exon 4 of APOE were amplified by PCR and sequenced by direct Sanger sequencing. A total of 18 variants were identified in 24 of these patients. The cascade screening in relatives of these 24 index patients allowed the identification and genetic characterization of additional 19 FH patients in Portugal. All alterations found have been previously reported, although only 11 had been functionally assessed. The search for large rearrangements was performed by Multiplex Ligation-dependent Probe Amplification (MLPA). In silico analysis was performed for all the variants found. III In order to access the effect of splicing mutations, RNA was isolated from patients’ blood with RNeasy® Mini Kit (Qiagen), after isolation of peripheral blood mononuclear cells, and then transcribed to cDNA. Regions of interest were amplified with specific primers designed to evaluate the effect on cDNA of two of the tree putative splicing variants found in LDLR gene. Specific detection of each transcript was accessed by an agarose gel and the fragments were sequenced by Sanger sequencing. Both alterations lead to skipping of an entire exon and create premature stop codons: c.1060+1G>A causes an inactivation of the donor site in intron 7 resulting in skipping of exon 7; the alteration in the last nucleotide of exon 16 (c.2389G>A) creates a new acceptor site causing the skipping of exon 16. The early genetic identification of a mutation, confirming the clinical diagnosis of FH, is very important, especially for young patients, since they can receive appropriate dietary and lifestyle advice and adequate therapeutic measures providing them longer and better lives.