Identification of pathogenic variants in the brazilian cohort with familial hypercholesterolemia using exon-targeted gene sequencing

Familial hypercholesterolemia (FH) is a monogenic disease characterized by high plasma low-density lipoprotein cholesterol (LDL-c) levels and increased risk of premature atherosclerotic cardiovascular disease. Mutations in FH-related genes account for 40% of FH cases worldwide. In this study, we aimed to assess the pathogenic variants in FH-related genes in the Brazilian FH cohort FHBGEP using exon-targeted gene sequencing (ETGS) strategy. FH patients (n = 210) were enrolled at five clinical sites and peripheral blood samples were obtained for laboratory testing and genomic DNA extraction. ETGS was performed using MiSeq platform (Illumina). To identify deleterious variants in LDLR, APOB, PCSK9, and LDLRAP1, the long-reads were subjected to Burrows-Wheeler Aligner (BWA) for alignment and mapping, followed by variant calling using Genome Analysis Toolkit (GATK) and ANNOVAR for variant annotation. The variants were further filtered using in-house custom scripts and classified according to the American College Medical Genetics and Genomics (ACMG) guidelines. A total of 174 variants were identified including 85 missense, 3 stop-gain, 9 splice-site, 6 InDel, and 71 in regulatory regions (3'UTR and 5'UTR). Fifty-two patients (24.7%) had 30 known pathogenic or likely pathogenic variants in FH-related genes according to the American College Medical and Genetics and Genomics guidelines. Fifty-three known variants were classified as benign, or likely benign and 87 known variants have shown uncertain significance. Four novel variants were discovered and classified as such due to their absence in existing databases. In conclusion, ETGS and in silico prediction studies are useful tools for screening deleterious variants and identification of novel variants in FH-related genes, they also contribute to the molecular diagnosis in the FHBGEP cohort.

Identification of pathogenic variants in the Brazilian cohort with Familial hypercholesterolemia using exon-targeted gene sequencing.

Familial hypercholesterolemia (FH) is a monogenic disease characterized by high plasma low-density lipoprotein cholesterol (LDL-c) levels and increased risk of premature atherosclerotic cardiovascular disease. Mutations in FH-related genes account for 40% of FH cases worldwide. In this study, we aimed to assess the pathogenic variants in FH-related genes in the Brazilian FH cohort FHBGEP using exon-targeted gene sequencing (ETGS) strategy. FH patients (n = 210) were enrolled at five clinical sites and peripheral blood samples were obtained for laboratory testing and genomic DNA extraction. ETGS was performed using MiSeq platform (Illumina). To identify deleterious variants in LDLR, APOB, PCSK9, and LDLRAP1, the long-reads were subjected to Burrows-Wheeler Aligner (BWA) for alignment and mapping, followed by variant calling using Genome Analysis Toolkit (GATK) and ANNOVAR for variant annotation. The variants were further filtered using in-house custom scripts and classified according to the American College Medical Genetics and Genomics (ACMG) guidelines. A total of 174 variants were identified including 85 missense, 3 stop-gain, 9 splice-site, 6 InDel, and 71 in regulatory regions (3'UTR and 5'UTR). Fifty-two patients (24.7%) had 30 known pathogenic or likely pathogenic variants in FH-related genes according to the American College Medical and Genetics and Genomics guidelines. Fifty-three known variants were classified as benign, or likely benign and 87 known variants have shown uncertain significance. Four novel variants were discovered and classified as such due to their absence in existing databases. In conclusion, ETGS and in silico prediction studies are useful tools for screening deleterious variants and identification of novel variants in FH-related genes, they also contribute to the molecular diagnosis in the FHBGEP cohort.

High-dose of potent statins may not be sufficient to achieve therapeutic goals of ldlc and non-hdl cholesterol in patients at very high cardiovascular risk

INTRODUCTION High-dose of potente statins are first-line and mandatory therapy to reduce LDL-c among very high cardiovascular risk patients. The Brazilian Guideline on Dyslipidemia and Prevention of Atherosclerosis of the Brazilian Society of Cardiology (SBC) recommends a target of LDL-c below 50mg/dL or non-HDL cholesterol (NHDLc) <80mg/dL. OBJECTIVE Evaluate the amount of very high-risk patients using the initial treatment recommended by the brazilian guideline that achieved the LDL-c and NHDLc therapeutic goals. METHODS This is a crosssectional observational study that included 1122 very high cardiovascular risk patients (significant atherosclerotic disease with or without clinical events or obstruction ≥ 50% in any arterial territory), treated in outpatients clinics between January and March of 2022, in a tertiary care hospital in Brazil, using atorvastatin 40 mg or 80 mg daily. Exclusion criteria were: use of atorvastatin in a dose lower than 40mg/daily or use of other statins. Data from the electronic medical record were collected regarding lipid profile, such as LDL-c and NHDLc, age and sex. RESULTS A total of 1122 patients were evaluated and 1012 were included. Mean age was 68.8 years (SD 9.2), 634 (62.4%) were men. Regarding statin use, 613 (60.6%) patients used atorvastatin 80mg/daily and 399 (39.4%) used atorvastatin 40mg/daily. Average LDL-c was 83.1mg/dL (SD 29.5) and NHDLc was 113.5mg/dL (SD 35). The mean TC was 152 mg/dL (SD 29.5), HDL-c was 39.5 (SD 11.8) and of TG was 154.9 (SD 85.6). Ninety two (9%) patients had LDL-c <50 mg/dL and 133 (13.14%) patients had NHDLc <80mg/dL. CONCLUSION The low amount of patients in this population that achieved LDL-c and NHDLc target shows that with high-intensity statins monotherapy may not be sufficient. These data suggest that in very high-risk patients, combined lipid-lowering therapy, in the initial phase, should be considered.

Genomics, epigenomics and pharmacogenomics of Familial Hypercholesterolemia (FHBGEP): A study protocol

BACKGROUND: Familial hypercholesterolemia (FH) is a genetic disease that affects millions of people worldwide. OBJECTIVES: The study protocol FHBGEP was design to investigate the main genomic, epigenomic, and pharmacogenomic factors associated with FH and polygenic hypercholesterolemia (PH). METHODS: FH patients will be enrolled at six research centers in Brazil. An exon-targeted gene strategy will be used to sequence a panel of 84 genes related to FH, PH, pharmacogenomics and coronary artery disease. Variants in coding and regulatory regions will be identified using a proposed variant discovery pipeline and classified according to the American College Medical Genetics guidelines. Functional effects of variants in FH-related genes will be investigated by in vitro studies using lymphocytes and cell lines (HepG2, HUVEC and HEK293FT), CRISPR/Cas9 mutagenesis, luciferase reporter assay and other technologies. Functional studies in silico, such as molecular docking, molecular dynamics, and conformational analysis, will be used to explore the impact of novel variants on protein structure and function. DNA methylation profile and differential expression of circulating non-coding RNAs (miRNAs and lncRNAs) will be analyzed in FH patients and normolipidemic subjects (control group). The influence of genomic and epigenomic factors on metabolic and inflammatory status will be analyzed in FH patients. Pharmacogenomic studies will be conducted to investigate the influence of genomic and epigenomic factors on response to statins in FH patients. SUMMARY: The FHBGEP protocol has the potential to elucidate the genetic basis and molecular mechanisms involved in the pathophysiology of FH and PH, particularly in the Brazilian population. This pioneering approach includes genomic, epigenomic and functional studies, which results will contribute to the improvement of the diagnosis, prognosis and personalized therapy of FH patients.

Genomics, epigenomics and pharmacogenomics of familial hypercholesterolemia (FHBGEP): A study protocol.

BACKGROUND: Familial hypercholesterolemia (FH) is a genetic disease that affects millions of people worldwide. OBJECTIVES: The study protocol FHBGEP was design to investigate the main genomic, epigenomic, and pharmacogenomic factors associated with FH and polygenic hypercholesterolemia (PH). METHODS: FH patients will be enrolled at six research centers in Brazil. An exon-targeted gene strategy will be used to sequence a panel of 84 genes related to FH, PH, pharmacogenomics and coronary artery disease. Variants in coding and regulatory regions will be identified using a proposed variant discovery pipeline and classified according to the American College Medical Genetics guidelines. Functional effects of variants in FH-related genes will be investigated by in vitro studies using lymphocytes and cell lines (HepG2, HUVEC and HEK293FT), CRISPR/Cas9 mutagenesis, luciferase reporter assay and other technologies. Functional studies in silico, such as molecular docking, molecular dynamics, and conformational analysis, will be used to explore the impact of novel variants on protein structure and function. DNA methylation profile and differential expression of circulating non-coding RNAs (miRNAs and lncRNAs) will be analyzed in FH patients and normolipidemic subjects (control group). The influence of genomic and epigenomic factors on metabolic and inflammatory status will be analyzed in FH patients. Pharmacogenomic studies will be conducted to investigate the influence of genomic and epigenomic factors on response to statins in FH patients. SUMMARY: The FHBGEP protocol has the potential to elucidate the genetic basis and molecular mechanisms involved in the pathophysiology of FH and PH, particularly in the Brazilian population. This pioneering approach includes genomic, epigenomic and functional studies, which results will contribute to the improvement of the diagnosis, prognosis and personalized therapy of FH patients.

Variante genética da doença do olho de peixe: relato de caso / Genetic variant of fisheye disease: case report

INTRODUÇÃO: Os níveis de colesterol ligados à lipoproteína de alta densidade (HDL-c) estão inversamente associados a elevado risco de doenças cardiovasculares (DCV). Dentre as causas de baixos níveis de HDL-c está a deficiência familiar de lecitina-colesterol aciltransferase (LCAT), enzima responsável pela esterificação do colesterol livre na superfície de lipoproteínas; sua deficiência leva ao rápido catabolismo das apolipoproteínas AI e AII, principais constituintes do HDL. Existem duas formas de déficit de LCAT: familiar, caracterizada por opacidades corneanas, anemia hemolítica e insuficiência renal; e doença olho de peixe, quando ocorre em homozigose, cursando com baixos níveis de HDL-c, nefropatia e opacidades em córneas. A doença coronariana geralmente não faz parte do fenótipoda síndrome do olho de peixe, mas pode ocorrer em algumas mutações, mesmo em heterozigotos. MÉTODOS: Realizada pesquisa em SciELO e PubMed sobre o tema e revisão de prontuário. RESULTADOS (RELATO): Paciente 35 anos, feminino, com antecedentes de nefrolitíase e depressão, encaminhada para hospital terciário de cardiologia para investigação de síncope. Exame físico inicial evidenciou arcos corneanos bilaterais, sem outros achados. Tilt Test positivo para síncope cardioinibitória. Para investigação de achado em exame físico foram solicitados exames laboratoriais, sendo evidenciados colesterol total = 61, HDL-c=05 e LDL-c=44. Realizou tomografia para avaliação de escore de cálcio, com resultado normal. Fundo de olho documentou arco corneano em ambos os olhos e Van Herick IV bilateral (figura 1), sugerindo doença do olho de peixe. Em teste genético foi evidenciado variante no gene da LCAT, com troca de Arginina por Histidina na posição 246 (Arg246His), previamente associada a HDL-c baixo em famílias italiana e portuguesa, levando a um fenótipo similar à doença do olho de peixe, em geral não associado a DCV. Paciente mantém seguimento ambulatorial, sem evidências clínicas ou subclínicas de aterosclerose ou eventos cardiovasculares até o momento, função renal e acuidade visual normais. CONCLUSÃO: A doença do olho de peixe e suas variantes são raras e caracterizam-se por níveis extremamente baixos de HDL-c. O impacto quanto à elevação do risco cardiovascular ainda não é consenso na literatura. O diagnóstico torna-se importante para a pesquisa de aterosclerose subclínica, identificação e tratamento complicações e pesquisa da doença em familiares. No caso relatado, paciente diagnosticada com variante de doença do olho de peixe, sem evidências de aterosclerose até o momento.

Exposição aos níveis de colesterol ao longo da vida e doença coronariana / Lifetime exposure to cholesterol evels and coronarysrtery disease disease

A doença aterosclerótica compreende amplo espectro de entidades clínicas com envolvimento genético e ambiental. A exposição ao longo da vida a níveis elevados de colesterol e de sua fração LDL determinam um limiar a partir do qual a doença aterosclerótica se desenvolve. Assim, nas formas genéticas de dislipidemias, como a hipercolesterolemia familiar, a idade do aparecimento da doença aterosclerótica vai depender da carga cumulativa de exposição aos níveis de LDL-colesterol, sendo tanto mais precoce quanto maiores os níveis de LDL-colesterol e a presença de fatores de risco adicionais, e mais tardia na ausência destes, e no sexo feminino. A prevenção ao longo da vida parece ser extremamente efetiva, e a avaliação individual com a implementação de medidas preventivas precoces e terapêuticas deve ser estimulada. Assim, parece lógico que reduções de colesterol, por mudanças no estilo de vida ou pelo uso de fármacos na adolescência e ao longo da vida apresentem inestimável benefício para a redução dos desfechos cardiovasculares na vida adulta

The atherosclerotic process comprises a broad spectrum of clinical entities, with genetic and environmental involvement. Lifetime exposure to high levels of cholesterol and LDL-cholesterol determine a trigger that can lead to the development of atherosclerotic disease. Therefore, in genetic forms of dyslipidemia, such as familial hypercholesterolemia, the age of onset of atherosclerotic disease will depend on the cumulative burden of exposure to LDL-cholesterol levels, being earlier with higher levels of LDL-cholesterol, the presence of other risk factors and later, in the absence of risk factors, and in females. Prevention throughout life appears to be extremely effective, and individual assessment, with the implementation of early preventive measures, should be encouraged. Thus, it seems logical that cholesterol reductions, changes in lifestyle, or the use of specific medications in adolescence and throughout life present inestimable benefit in reducing cardiovascular outcomes in adulthood

Controle lipídico total: além do LDL-colesterol / Total lipidic control nesides: LDL-cholesterol

A doença aterosclerótica trata-se de processo multifatorial que tem início na infância, mas que geralmente se manifesta mais tarde na vida. O desenvolvimento desta envolve fatores genéticos, bem como fatores de risco adquiridos e modificáveis, dentre eles o tabagismo, as dislipidemias, a hipertensão arterial e o diabetes mellitus. A redução do LDL-colesterol demonstrou grande benefício na redução de eventos e na mortalidade cardiovascular, bem como na mortalidade por todas as causas, principalmente com o uso das estatinas. No entanto, é inquestionável que essas medicações estão longe de eliminar todo o risco de eventos cardiovasculares, ou seja, existe um risco residual. Na tentativa de diminuir ainda mais o risco cardiovascular, devemos tratar outros fatores envolvidos na gênese da aterosclerose. O tratamento da hipertrigliceridemia, do HDL-colesterol baixo, do colesterol não HDL, da relação ApoB/ApoA são alguns dos exemplos mencionados neste artigo. Novos tratamentos estão sendo desenvolvidos na tentativa de diminuir o risco residual, exemplo são os inibidores do PCSK-9, os inibidores da MTP e os inibidores da fosfolipase A2. Todos ainda em fase de testes em humanos, mas que poderão ser armas de grande utilidade no nosso arsenal terapêutico futuro.

Cardiovascular atherosclerotic disease is a multifactorial process that begins in childhood but it usually manifests later in life. Its development involves genetic factors as well as acquired and modifiable risk factors, including smoking, dyslipidemia, hypertension and diabetes mellitus. The reduction of LDL-cholesterol showed great benefit in preventing cardiovascular events and mortality and deaths from all causes, especially with the use of statins. However, it is unquestionable that these medications are far from obtaining total elimination of the risk of cardiovascular events, in other words, there is a residual risk. In an attempt to further reduce cardiovascular risk, we must address other factors involved in atherogenesis. Treatment of hypertriglyceridemia, low HDL-cholesterol, non HDL-cholesterol and the ApoB/ApoA ratio are some of the examples mentioned in this article.New treatments are being developed in an attempt to reduce the residual risk, such as PCSK-9, MTP and phospholipase A2 inhibitors. All still in human trials therefore they might become very useful weapons in the future.

Avaliação do risco coronário: importância das apolipoproteínas / Coronary risk evaluation: importance of apolipoproteins

Embora muitos avanços tenham sido obtidos na prevenção da aterosclerose, esta doença, com suas variadas manifestações, permanece como a maior causa de morte nos países ocidentais. A principal forma de lutar contra a aterosclerose envolve o combate aos seus fatores de risco de há muito conhecidos. Entretanto, novos conceitos têm surgido e entre estes se destaca o papel das apolipoproteínas como marcadores do risco coronário. Entre suas variadas funções, as proteínas que fazem parte das lipoproteínas mostraram que a determinação de seus níveis sangüíneos pode acrescentar informações sobre a situação individual de risco, particularmente com as dosagens das apos B e A-1 e da relação entre elas. Este artigo revisa os dados disponíveis sobre o papel fisiológico e de indicador de risco dessas apolipoproteínas, sugerindo o que já fazem alguns autores que defendem a idéia da incorporação da determinação rotineira dessas proteínas ao perfil lipídico para avaliação do risco cardiovascular.

Efeitos pleiotrópicos com reduções equivalentes do LDL-colesterol: estudo comparativo entre sinvastatina e associação sinvastatina/ezetimiba / Pleiotropic effects with equivalent LDL-cholesterol reduction: comparative study between simvastatin and simvastatin/ezetimibe co-administration

A associação de uma estatina com ezetimiba é tão eficaz quanto altas doses da mesma estatina na redução do LDL-colesterol. Objetivo: comparar a ação de dois esquemas de tratamento que obtêm reduções equivalentes de LDL-colesterol (sinvastatina 80 mg ao dia e associação sinvastatina 10mg/ezetimiba 10 mg ao dia), sobre os efeitos pleiotrópicos: inflamação, função endotelial e oxidação da LDL. Métodos: estudamos 23 pacientes em cross-over. Resultados: os dois grupos apresentaram melhora da função endotelial e PCR-us, sem diferenças entre os tratamentos. Houve aumento na concentração do anticorpo anti-LDL- apenas no grupo sinvastatina.

The co-administration of a statin with ezetimibe is as effective as high doses of the same statin in the reduction of the LDL-cholesterol. Objective: compare the effectiveness of these two different treatments that obtain equivalent reductions of LDL-cholesterol (simvastatin 80 mg once a day and co-administration of simvastatin 10 mg once a day and ezetimibe 10 mg once a day), about pleiotropic effects: inflammation, endothelial function and LDL oxidation. Methods: we have studied 23 patients in cross-over. Results: the two groups presented improvement in endothelial function and high-sensitive C reactive protein, with no significative difference in any of the two treatments. The anti-LDL- antibodies concentration was increased only in the simvastatin group.