Browsing by Author "Menezes, J."
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- Functional characterization of LDL receptor gene variants found in patients with clinical diagnosis of familial hypercholesterolaemiaPublication . Alves, A.C.; Graça, R.; Abrantes, L.; Medeiros, A.M.; Queirós, R.; Menezes, J.; Romão, L.; Bourbon, M.Aim: Familial Hypercholesterolaemia (FH) is an autosomal disorder of lipid metabolism presenting increased cardiovascular risk due to lifelong exposure to high LDL levels. LDLR mutations are the cause of disease in about 90% of the cases, but proof of pathogenicity has only been obtained for about 10% of the variants. The lack of functional characterization of variants found in patients with a clinical diagnosis of FH makes it difficult to reach a definite FH diagnosis. In the Portuguese Familial Hypercholesterolemia Study (PFHS), 142LDLR alterations were found in 861 index patients and their relatives up to date. Until now, 82 of these alterations have already been proved to be mutations causing disease or are established null alleles and 15 were classified by in vitro studies as non-pathogenic. The remaining 38 variants, 21 of unknown significance (VUS), found in 102 index cases have not been yet functionally characterized.
- Regulatory Variants In LDLR And PCSK9 Promoters And 5'UTRs: Investigating The impact In Familial HypercholesterolaemiaPublication . Graça, R.; Menezes, J.; Fernandes, R.; Alves, A.C.; Romão, L.; Bourbon, M.Background and Aims: Familial Hypercholesterolaemia (FH) is a genetic disorder of lipid metabolism caused by pathogenic variants in LDLR, APOB, and PCSK9. While diagnostic efforts traditionally focus on coding variants, non-coding regions, such as promoters and 5'UTRs, remain understudied despite their importance. This work aims to characterise 100 variants in the promotor/5'UTR of LDLR and PCSK9. Methods: The promotor/5'UTR sequences of LDLR and PCSK9 were cloned by SOEing PCR upstream of the Firefly luciferase coding region in the pGL4.10. For LDLR, sequence from c.-319 to the initiation codon was retrieved from literature, while for PCSK9, sequence from c.-650 to the initiation codon was confirmed using a 5'-RACE strategy. The resulting constructs (LDLR_pGL4-WT and PCSK9_pGL4-WT) were subsequently modified through site-directed mutagenesis. LDLR and PCSK9 variants were transfected into CHO-ldlA7 and Huh7 cells, respectively. Cells were cultured in different cholesterol depletion states, and luciferase activity measured using a Dual-Luciferase Reporter Assay System. Results: Compared to their respective wild-type constructs, LDLR and PCSK9 variants displayed a diverse range of phenotypic effects, with statistically significant increases or decreases in promoter activity. These variations can differently impact the FH phenotype and hold significant implications for disease management and therapeutic strategies, as increases or decreases in promoter activity in the two genes have distinctly opposing effects on LDL-C levels. Moreover, as far as we know, this is the first experimental work defining the PCSK9 5’ UTR region. Conclusions: This study provides novel insights into the functional impact of LDLR and PCSK9 promoter/5'UTR variants on gene expression and their potential contributions to the FH phenotype. Importantly, these findings underscore the critical role of functional studies in variant classification, particularly for non-coding regions, which remain underrepresented in genetic diagnostics. By elucidating how these variants influence LDL-C levels through altered promoter activity, this work highlights their relevance in refining FH diagnosis and tailoring patient management strategies.