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- Unveiling the Role of APOB Variants in Familial Hypercholesterolemia: Functional InsightsPublication . Ferreira, Maria Simões; Ramos, Diana; Rato, Inês; Jannes, Cinthia E.; Larrea-Sebal, Asier; Martín, César; Bourbon, Mafalda; Alves, Ana CatarinaFamilial hypercholesterolemia (FH) is a condition characterized by increased LDL cholesterol levels with APOB variants accounting for about 5-10% of FH cases. However, variants in this gene may be more common than initially estimated since the entire APOB gene has only recently started to be sequenced. Although most of the alterations are missense, nonsense variants and small indels in exon 29 were also identified in individuals with FH phenotype and can be the cause of disease. This work aimed to characterize APOB variants identified in individuals with clinical diagnosis of FH. Moreover, we intended to do an overview of the APOB variants presenting functional studies. PubMed repository was consulted to collect publications regarding functional characterization of APOB variants. For variant characterization, LDL was isolated through sequential ultracentrifugation. ED-LDLR fragments purified from HEK293 cells were incubated with the different APOB variants and antibodies, to determine apoB affinity for LDLR by ELISA assay. CHO-ldlA7 cells were transfected with wt LDLR plasmid and incubated with FITC-labeled LDL to determine LDL binding and uptake by flow cytometry. In the literature there are 23 APOB variants with functional studies, six of which characterized by our group. Fourteen variants affecting apoB normal function; the remaining results presenting normal apoB function. Recently we characterized 8 more variants: p.(Ala1393Val), p.(Asp1456Asn), p.(Met2042Thr), p.(Asp2213del), p.(Ile3374Thr), p.(Val4295Leu) and p.(Arg4519Thr) that do not appear to impact apoB's binding to the LDLR; p.(Gln4316*) demonstrated reduced affinity for the LDL receptor. Functional studies play a critical role in assessing the pathogenicity of genetic variants and are among the key criteria for variant classification. These in-depth analyses confirm clinical diagnosis and provide essential insights for developing personalized treatment strategies. In the future, we aim to increase the number of studied variants, starting with 15 more variants from the Portuguese FH Study.
- 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.