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Functional genomics in familial dyslipidaemia
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Dyslipidaemia is defined as an abnormal deviation of plasma lipid levels, being a significant risk factor for cardiovascular disease, which can be due to both genetic and environmental factors. There are several familial dyslipidaemias, and their occurrence is regulated by multiple genes. Familial hypercholesterolaemia (FH) is one of those dyslipidaemias, being the most common inherited life-threatening disorder of the lipid metabolism, affecting around 1:300 individuals. This disorder is characterised by increased levels of total and LDL plasma cholesterol, which is associated with premature atherosclerosis and coronary heart disease. The FH phenotype has a considerable genetic heterogeneity and phenotypic variability, depending on both the LDL receptor activity and lifestyle habits. So far, about 3,500 unique LDLR variants have been described, most of them lacking functional evidence proving their effect on the LDLR function. The high number of LDLR variants described as variants of uncertain significance (VUS) is the largest obstacle to achieve a definitive FH diagnosis, being early diagnosis and treatment the key to reduce the cumulative life-long cardiovascular burden of patients with FH.
Being a correct diagnosis one of the most important steps for FH patients’ identification this work concentrated in achieving a better diagnosis for FH patients by improving variants functional characterisation. Another aim was to unravel the genetic background of phenotype-positive genotype-negative patients in order to provide a more personalised medicine for these patients.
In the initial stage of this work, the combined efforts for the functional characterisation of 28 rare LDLR variants found in the Portuguese population (and other countries worldwide) allowed the classification reassignment of 15 variants, from VUS to likely benign (1), benign (3), likely pathogenic (10) or pathogenic (1). The variants were analysed by flow cytometry to assess their individual function and the results showed a large spectrum of functional levels, indicating that the impact of variants is not uniform across the LDLR gene. This methodology is deemed as the gold standard for functional characterisation and recommended by the FH Variant Curation Expert Panel from the Clinical Genome Resource. These results allowed 82 Portuguese patients to receive a definitive FH diagnosis, as well as many other FH patients with the same variants scattered across the 21 different countries where the same variants have been described.
Following the first stage of this work, the efforts were focused on the development of a high-throughput cell-based methodology to identify disruptive variants in the LDLR gene, with the objective of providing a fast and reliable method for variant characterisation. This assay showed to be a time- and cost-effective method of profiling rare variants, helping to differentiate between rare variants that are clinically significant and those that are benign. Two sets of variants were characterised: 1 – control set, consisting of 22 previously characterised variants as well as variants that achieve a pathogenic and benign classification without functional studies (n=5), where no variants were misclassified; 2 – test set, with 19 newly functionally characterised variants: 11 displaying abnormal function, five displaying normal function, and three remaining unclassified. This work generated a valuable resource for systematic functional characterisation of LDLR variants, helping to solve one of the major issues to achieve a definitive FH diagnosis. Large scale up of this method is now possible.
This project also assessed the impact of rare variants from an array of 112 genes on a FH-negative cohort gathered from the Portuguese FH study. From the targeted sequencing of 185 variant-negative individuals and subsequent cascade family screening, it was possible to determine a definitive or possible genetic aetiology of the dyslipidaemia for 14 patients. The obtained data uncovered a high prevalence of rare variants in genes associated with sitosterolaemia and hypertriglyceridaemia, namely ABCG5, ABCG8 and GPD1 genes. Heterozygous variants in these genes are linked to increases of LDL cholesterol levels of up to 71.38, 76.11 and 65.96 mg/dL, respectively, compared to normal controls. From the analysis of this cohort, it was described the second case of sitosterolaemia in Portugal. Finally, in three unrelated families it was identified a variant that largely co-segregates with the phenotype in each family, in a new potential FH gene. However, further studies and supportive evidence are still required to establish a direct association to the FH phenotype and pathogenesis.
Overall, this work makes a significant contribution to the field of dyslipidaemias and FH, by introducing new data and fresh perspectives to various domains within it. It contributed for the functional characterisation of over 40 rare LDLR variants, which can allow a more personalised choice of the lipid-lowering treatment and dyslipidaemia management, ultimately improving patients’ prognosis. Moreover, a new high-throughput cell-based methodology that generates a useful resource for systematic functional characterisation of rare LDLR variants has been developed. This method has the potential to quickly profile a significant number of variants of uncertain significance, addressing a major gap in the diagnosis of FH. Additionally, this research shed light on the genetic dynamics of FH-positive and FH-negative individuals, highlighting the importance of accurate identification of dyslipidaemia's aetiology and introduces a potential candidate FH gene, providing a new avenue for future investigation.
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Familial hypercholesterolaemia LDLR Functional studies ACMG variant classification Definitive diagnosis