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- Base genética da hipercolesterolemia familiarPublication . dos Santos Alves, Ana Catarina; Bourbon, Mafalda; Farinha, Carlos[PT] A Hipercolesterolemia Familiar (FH) é uma doença autossómica dominante caracterizada clinicamente por um aumento dos níveis de colesterol LDL no plasma, conduzindo à sua acumulação principalmente nos tendões (xantomas tendinosos) e nas artérias. Devido à acumulação de lípidos nas artérias, estes indivíduos desenvolvem aterosclerose muito cedo, tendo eventos cardiovasculares prematuramente. A FH apresenta uma frequência de 1/500, na forma heterozigótica, na maioria das populações europeias. Em Portugal estima-se que existam cerca de 20 000 casos de FH. A identificação clínica da FH é possível, mas apenas o diagnóstico molecular da FH permite a correta identificação da doença, fundamentando a instituição de terapêutica farmacológica mais agressiva e/ou precoce, com a consequente redução do risco cardiovascular nos indivíduos afetados. Geneticamente esta patologia caracteriza-se por mutações em três genes: LDLR, APOB e PCSK9. No entanto, a percentagem de indivíduos identificados molecularmente situa-se entre 30-80% dependendo da população estudada, uma vez que em Portugal apenas 40% dos doentes com diagnóstico clínico de FH apresentam uma mutação causadora de doença num dos três genes associados à FH. O Estudo Português de Hipercolesterolemia Familiar (EPHF) tem como objetivos a realização de um estudo epidemiológico para a determinação da prevalência e distribuição da FH em Portugal, tendo implementado o estudo molecular desta doença. Para tal, a caracterização funcional de alterações encontradas em genes associados à FH é de extrema importância pois permite obter um diagnóstico definitivo de FH. O objetivo deste trabalho doutoral era identificar e caracterizar a causa genética da hipercolesterolemia em famílias sem mutação identificável nos genes LDLR e APOB pelas metodologias usualmente utilizadas. A primeira parte do presente foi dedicada à reorganização do diagnóstico molecular da FH e à pesquisa de mutações num novo gene – PCSK9 –, à pesquisa de grandes rearranjos através da técnica de MLPA, bem como ao estudo dos genes APOB e LDLRAP1 através de duas abordagens diferentes da técnica de pirosequenciação. No total foram estudados no EPHF 642 casos índex (CI) (250 crianças e 392 adultos), tendo sido identificada uma alteração em 294 indivíduos. A reorganização do EPHF permitiu identificar 6 grandes rearranjos através da técnica de MLPA, identificando deste modo mais 19 indivíduos e 4 alterações no gene PCSK9 em 5 CI. Através da técnica de pirosequenciação, identificaram-se 10 novas alterações potencialmente patogénicas no gene APOB em 10 indivíduos e 4 alterações no gene LDLRAP1 em 4 CI. A segunda parte do trabalho incluído nesta tese centrou-se na análise funcional de alterações cuja patogenicidade era desconhecida, uma vez que dos 294 CI com uma alteração apenas 192 CI apresentam uma mutação comprovadamente patogénica. Desta forma, foram analisadas funcionalmente para o gene LDLR 11 alterações de splicing, uma alteração no promotor e 4 alterações pontuais. Verificou-se que 8 alterações de splicing e 3 alterações missense eram patogénicas, e que 3 alterações de splicing e 1 alteração missense não originavam doença. No gene APOB foram estudadas funcionalmente 4 alterações, das quais apenas 2 destas são patogénicas. Estas mutações foram as primeiras descritas fora da região consensus de ligação do ligando ao LDLR postulada anteriormente. Na impossibilidade da caracterização funcional de todas as alterações cujo estudo não tinha sido realizado, foi desenvolvida uma classificação com base em 6 programas bioinformáticos para as alterações missense e 3 programas para as alterações de splicing. Com base nesta classificação, identificaram-se 435 CI (175 CI e 260 familiares) com diagnóstico definitivo de FH, e 196 indivíduos (116 CI e 80 familiares) apresentavam um diagnóstico provável, 79 um diagnóstico possível e 46 neutro. Verificou-se que 594 indivíduos apresentavam uma alteração no gene LDLR, 30 indivíduos têm uma alteração no gene APOB e 7 indivíduos no gene PCSK9. A terceira parte deste trabalho doutoral teve como objetivo correlacionar o genótipo vs com o fenótipo de todos os CI referenciados ao EPHF, tendo por base os diferentes diagnósticos moleculares que foram obtidos através da classificação das alterações com e sem estudos funcionais. A comparação dos fenótipos apresentados por crianças e adultos com diferentes tipos de mutação revelou que, embora em idade pediátrica o fenótipo não dependa do tipo de mutação, na idade adulta já existe uma clara diferença de fenótipos entre portadores de mutações nulas e missense. Esta observação reforça a importância do diagnóstico molecular precoce de modo a estratificar o risco cardiovascular no grupo pediátrico, uma vez que as crianças com mutações nulas apresentam um maior risco cardiovascular, necessitando por isso de um aconselhamento especializado e implementação ainda mais precoce de terapêuticas hipolipemiantes. A última parte deste trabalho focou-se na realização do estudo de sequenciação de exoma a 5 CI com diagnóstico clínico de FH, mas sem uma mutação identificada num dos 3 genes associados à FH. Uma vez que a sequenciação de exoma origina uma quantidade elevada de dados por amostra, de modo a restringir a análise, foi realizada uma seleção de 49 genes associados ao metabolismo lipídico, tendo-se verificado a existência de 20 alterações distribuídas por 17 genes. Através deste estudo, identificaram-se 2 alterações no gene FLT1 em 2 CI não relacionados e 1 alteração no gene SORT1 noutro CI que cossegregam com a hipercolesterolemia na família. Os estudos funcionais das alterações encontradas nestes genes serão importantes para classificar as mesmas quanto à sua patogenicidade. No caso de se comprovar que alguma destas alterações é funcional, esta será a primeira evidência de um quarto gene associado a FH. Uma vez que a FH se encontra sub-diagnosticada em Portugal, o diagnóstico e aconselhamento genético da FH são importantes para a correta perceção e prevenção do risco familiar de DCV. Nas crianças e adolescentes, o diagnóstico genético é ainda mais importante, uma vez que se sabe que o risco cardiovascular é elevado, mas evitável, se medidas preventivas forem colocadas em prática. O futuro passa pela prevenção, em vez da resolução tardia das complicações cardiovasculares inerentes a esta patologia.
- Biochemical and molecular characterisation of the dyslipidaemia in PortugalPublication . Costa, Cibelle Neiva Cavalcanti Mariano da; Bourbon, Mafalda; Antunes, MaríliaABSTRACT: Dyslipidaemia is one of the major modifiable independent risk factors for cardiovascular disease (CVD), with both genetic and environmental determinants. Although genetic risk factors are considered as non-modifiable, their CVD-associated risk can be prevented if early identified. The correct and early identification of dyslipidaemia is important for a better patient management and could definitely contribute to CVD prevention. This thesis intended the most complete characterisation of the dyslipidaemia in the Portuguese population, both biochemically and molecularly. Reference values based on population-specific percentiles for lipid and lipoprotein biomarkers were provided for the first time in the Portuguese population, namely total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), apolipoprotein A1 (apoA1), apolipoprotein B (apoB), small, dense LDL-C (sdLDL-C), lipoprotein(a) [Lp(a)], as well apoB/apoA1 and sdLDL-C/LDL-C ratios, and non-HDL-C and remnant cholesterol. To our knowledge, the sdLDL-C percentiles were the first to be established in an European population. The percentiles were estimated through a rigorous methodology and compared with other population percentiles by a very visual and feasible method, showing relevant differences. These newly determined reference values for lipid biomarkers were then used to characterise the dyslipidaemia in our population, and can now be used in the clinic for a better patient care and management. More than cholesterol per se, our study highlighted apoB and sdLDL-C as important biomarkers to be used in dyslipidaemia evaluation. Individuals presenting extreme phenotypes were further investigated to assess possible monogenic causes, and three individuals were found to have familial hypercholesterolemia (FH), the most common genetic dyslipidaemia and one of the most common disorders that confer an increased cardiovascular risk. Finally, in an attempt to explore the causes for the FH phenotype, a polygenic risk score was validated for the first time in the Portuguese population. A total of 289 index cases were identified with monogenic FH and other causes for their dyslipidaemia, and also 100 were identified with polygenic hypercholesterolaemia, representing 53.21% of the cohort. From the monogenic causes, 91.35% have a mutation in LDLR, 4.84% in APOB, 1.04% in PCSK9 and 2.08% had mutations in phenocopies genes (LIPA, APOE, ALB), suggesting that all those monogenic and polygenic causes should be always investigated for a better patient identification. This study provided the most complete characterisation of the dyslipidaemia in the Portuguese population, and important evidences for dyslipidaemia evaluation has been produced. The results obtained have application, not only for Portugal or a south European populations, but also might have an worldwide utility for the dyslipidaemia assessment. Together, the results obtained provide useful information on an important cardiovascular risk factor and should help to tackle and identify at risk situations that need urgent measures.
- Development and Validation of Screening Methods Applied to Familial Hypercholesterolemia DiagnosisPublication . Albuquerque, João; Antunes, Marília; Antunes, Marília; Bourbon, Mafalda; Soares, RaquelFamilial hypercholesterolemia (FH) is an inherited disorder of lipid metabolism, characterized by increased low density lipoprotein cholesterol (LDLc) levels. If untreated, the severe dyslipidemia from birth leads to the early development of atherosclerosis, representing a major risk factor for cardiovascular disease (CVD). The early diagnosis of FH is associated with a signi cant reduction in CVD risk, supporting the introduction of risk mitigation strategies, such as cascade screening of rst degree relatives, and adequate lipid lowering therapy (LLT) as precociously as possible. The importance of genetic testing is emphasized by evidence that individuals with a con rmed pathogenic variant possess a signi cant increase in the risk of CVD when compared to subjects with FH-like phenotype for whom a causative variant is not detected. Nevertheless, molecular testing is still not available as a rst line diagnosis tool, and previous selection and strati cation of subjects to undergo this procedure should be made. Currently used clinical criteria, typically based on LDLc levels, family history of hypercholesterolemia and/ or premature CVD and presence of physical signs like tendon xanthomas, present the limitation of retaining a high number of false positive cases. This may constitute a heavy burden in terms of healthcare costs, and limits the access to the genetic study of a larger universe of true FH cases. The main purpose of this work was to develop alternative classi cation methods for FH diagnosis, based on di erent biochemical and clinical indicators, with improved ability to screen for FH cases in comparison to traditional clinical criteria. The metrics used for comparison range from the areas under the receiver operating characteristics (AUROC) and precision-recall (AUPRC) curves, to several operating characteristics (OC), to agreement tests, among others
- Tackling the molecular basis of lipid metabolism: from candidate genes testing in a disease cohort to multi-omics approaches in unselected populationsPublication . Rossi, Niccolò; Bourbon, Mafalda; Falchi, MárioDyslipidemia, broadly defined as an unhealthy deviation of plasma lipid levels, is a well-known heritable risk factor for c ardiovascular diseases (CVD), the first cause of death worldwide. Uncovering the genetic basis of p lasma lipids is, therefore, fundamental for CVD prevention and treatment. In this work, I tackled dyslipidemia from two different perspectives, namely genetics of severe dyslipidemia in a disease cohort and multi-omics of plasma lipids variation in unselected populations. First, I aimed to investigate if mutations in genes involved in miscellaneous monogenic dyslipidemia can mimic a Familial Hypercholesterolemia-like phenotype. By knocking-down dyslipidemia genes in cultured cells, I observed that sitosterolemia and hypertriglyceridemia causing genes are negative regulators o f LDL-uptake in vitro. Targeted sequencing of 1 85 FH mutation-negative individuals from the Portuguese FH study and subsequent cascade family screening for c andidate pathogenic variants, highlighted nine variants in ABCG5, ABCG8 and GPD1 co-segregating with the FH-phenotype. Mutations in these three genes, in heterozygosity, were associated with increased plasma LDL-cholesterol (LDL-C) as compared to the normal population (β = +71.38±9.57, +76.11±10.14 and +65.96±8.77 mg/dL, respectively). Rare genetic variants underlying extreme dyslipidemia tend to be conserved across ethnic groups. Instead, the study of rare variations underlying non-monogenic dyslipidemia in multi-ethnic populations remains challenging. Here, I looked for rare single nucleotide variants, individually or in aggregate, associated with plasma LDL-C from whole-genome sequencing data in 1,751 participants f rom the TwinsUK c ohort and replicated my findings in 2,587 individuals from the Qatar Genome Programme. I identified a conserved locus located upstream the KCNJ2 gene associated with LDL-C levels, at both single and aggregate variants levels in the two cohorts, and with myocardial infarction risk in TwinsUK. Loci identified by association studies have the potential to reveal novel genes and pathways involved in dyslipidemia biology. However, individual genes do not work alone, but rather interact with one another and jointly affect human health. I constructed gene co-expression networks based on RNA sequencing data generated from subcutaneous adipose and skin tissues, and lymphoblastoid cell lines from 856 subjects from the TwinsUK cohort. First, by testing the enrichment of co-expression modules for l ipid-related gene ontologies and GWAS hits, I defined a lipid functional gene module. Within this module, the expression level of the long non-coding RNA LINC00263 and transcription factor Srebf1, a key player in adipogenesis, were found to be highly correlated (Pearson’s ρ = 0.62; P = 3.71x10 -81) . In addition, I observed that LINC00263 predicted interactors are specifically expressed in adipocytes and are enriched for lipid-related pathways. Thus, I propose LINC00263 as a novel candidate lipid regulator in subcutaneous adipose tissue. Together, the results presented in this thesis provide new insights into dyslipidemia complex aetiology both at the genomic and transcriptomic level, and improve CVD risk assessment and prevention.
- uORF-mediated translational regulation of PERK: implications for cell homeostasis and human diseasePublication . Fernandes, Rafael; Romão, Luísa; Bourbon, MafaldaUpstream open reading frames (uORFs) are cis-acting elements located within the 5’ leader sequence (or 5’ untranslated region; 5’UTR) of transcripts that can regulate translation of the correspondent main open reading frame (mORF). During basal conditions, uORFs are typically considered to be repressors of downstream translation, as they can impede the ribosomes to access the mORF or even induce mRNA degradation by the nonsense-mediated mRNA decay (NMD) pathway. However, during stress conditions, phosphorylation of the eukaryotic initiation factor 2α (eIF2α) allows the expression of several stress-responsive proteins through uORF-mediated mechanisms, while global mRNA translation is inhibited. During endoplasmic reticulum (ER) stress, for instance, the accumulation of unfolded proteins leads to activation of the ER-resident PKR-like ER kinase (PERK) that phosphorylates eIF2α as part of the stress-protective mechanisms of the unfolded protein response (UPR) and the integrated stress response (ISR). This results in the selective uORF-mediated translation of downstream effectors, like the activating transcription factor 4 (ATF4), the CCAAT-enhancer-binding protein homologous protein (CHOP) and the growth arrest and DNA damage-inducible protein 34 (GADD34), which drive stress resolution or, in the case of a prolonged stress, cell death. The dual role of PERK in regulating cell fate has been reported to be involved in the outcome of several human diseases, including diabetes, neurodegenerative disorders and cancer. Moreover, mutations in the EIF2AK3 gene that encodes PERK have been implicated in the development of the rare genetic disease, Wolcott-Rallison Syndrome (WRS). Interestingly, data from ribosome-profiling (Ribo-seq) studies suggested the existence of uORFs within PERK 5’UTR, which could be involved in the regulation of PERK expression. In this work, we aimed to study the translational regulatory role of five AUG- and three non-AUG-uORFs identified in the PERK 5’UTR and assess its impact in cell homeostasis and human disease. While uORF2 and the non-AUG-uORFs 5, 6 and 7 do not seem to have a significant regulatory role, uORF1, uORF3, uORF4 and uORF8 together present a strong repressive effect over mORF translation in basal conditions, possibly by providing a barrier to the scanning ribosomes and precluding translation reinitiation at the mORF, without affecting the PERK mRNA levels. Curiously, we found that when we induce PERK overexpression, it leads to the spontaneous activation of a portion of PERK in the absence of any stress stimulus, possibly highlighting the biological relevance of its uORF-mediated translational regulation in maintaining its physiological basal levels. Conversely, during stress conditions, increased bypass of uORF1 results in a modest degree of translational de-repression, which may help to counterbalance the increased rate of PERK protein turnover observed in these conditions. We also found that alteration of the PERK uORFs by mutations found in WRS patients modify PERK expression, providing a possible link with the disease phenotype. Finally, we tested the impact of PERK unbalanced expression in the viability of HCT116 cells but, at least in our experimental conditions, no differences were found. Altogether, we provide a new example of a transcript containing uORFs that fine-tune mORF translation. Moreover, we highlight the importance of including 5’UTRs, like the one of PERK, in the screening of stress-related mutations and the necessity of functional studies to assess their relevance in the pathogenesis of human diseases. This may provide vital information for the development of new therapeutic strategies.