Advances in diagnosis and treatment of familial hypercholesterolemia / 南方医科大学学报

Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease caused by abnormal lipoprotein metabolism. Patients with FH have a significantly increased risk of coronary artery disease (CAD) due to long-term exposure to high levels of low-density lipoprotein (LDL). The diagnosis of FH relies heavily on gene detection, and examination of LDL receptor (LDLR) function is of great significance in its treatment. This review summarizes the current advances in the screening, diagnosis, and treatment of FH and functional analysis of LDLR gene mutations.

An Early Onset of Myocardial Infarction Patient with Syphilis and Familial Hypercholesterolemia / 罕见病研究

Syphilis may affect the cardiovascular system, in which coronary arteries are less commonly involved. Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease with elevated low-density lipoprotein-cholesterol (LDL-C) levels due to impaired LDL-C clearance. We report a young male patient with syphilis and FH. The clinical manifestations were acute myocardial infarction and high LDL-C levels. Coronary angiography and intracoronary imaging showed multiple aneurysmal ectasia and stenosis. A drug-eluting stent was implemented when recurrent restenosis occurred after two percutaneous coronary drug-eluting balloon angioplasties.

Familial Hypercholesterolemia / 罕见病研究

Familial hypercholesterolemia (FH) is a group of autosomal co-dominant genetic diseases mainly characterized by abnormal low-density lipoprotein related metabolism. It is one of the most common inherited diseases in children and one of the most serious lipid metabolism diseases which results in various life-threatening cardiovascular diseases and the complications. In recent years, the treatment protocols for FH have diversified thanks to the deeper understanding of the disease in China and abroad and the development of new lipid-lowering drugs. However, the current awareness and diagnosis rate of FH are very low. The treatment of the disease is much inadequate. This paper summarizes the clinical characteristics, diagnosis, screening strategy, and treatment of FH hoping to enhance the understanding and awareness of the disease in the society.

A Case Report of Homozygous Familial Hypercholesterolemia Liver Transplantation / 罕见病研究

Homozygous familial hypercholesterolemia (HoFH) is a rare and serious autosomal genetic metabolic disease. Patients without intervention often die younger than 30 years old from early atherosclerotic cardiovascular disease (ASCVD)incurred by extremely high levels of low-density lipoprotein cholesterol (LDL-C). We present a case of HoFH, a child with compound heterozygous mutation in this study. The effect of conventional lipid-lowering therapy through diet control and lipid-lowering drugs was unsatisfactory. The blood-lipid purification proves effective but has poor compliance and difficult to maintain for a longer time. The patient received orthotopic liver transplantation and had been followed for 2 years, with the patient shows normal LDL-C, well growth and development. We hope the case will provide the clinician with better understanding of the diagnosis and treatment of the rare disease of HoFH.

Clinical research advances of liver transplantation for patients with familial hypercholesterolemia / 中华器官移植杂志

Familial hypercholesterolemia(FH)is an autosomal dominant disease.Homozygous FH patients tend to have a high incidence of severe arteriosclerotic cardiovascular disease during adolescence and die at ages of 20-30.Liver transplantation(LT)may correct the dysfunction of LDL receptor on hepatocytes, restore normal lipoprotein metabolism, arrest disease progression and improving patient outcomes.However, due to a great rarity of LT for FH, domestic transplantation centers lack the relevant clinical experiences.This review summarized some important issues of FH patients undergoing LT, such as indications, timing, donor sources, efficacies, complications and reusing diseased liver.The goal was to provide practical references for managing FH.

Criterios predictores en el diagnóstico de la hipercolesterolemia familiar / Predictive criteria in the diagnosis of familial hypercholesterolemia

Introducción: La hipercolesterolemia familiar es una enfermedad con alta prevalencia, no tratada acorta la esperanza de vida, por lo que el diagnóstico a edades tempranas resulta fundamental. Las pruebas genéticas constituyen el gold standard para el diagnóstico de hipercolesterolemia familiar, sin embargo, la no disponibilidad del test genético no debe constituir un impedimento para la adecuada conducta en estos casos. Objetivo: Identificar criterios clínicos predictores en el diagnóstico por pesquisa de la hipercolesterolemia familiar. Métodos: Se realizó un estudio descriptivo prospectivo a partir de una muestra de 393 pacientes (casos índices) de HF en el Hospital Clínico Quirúrgico Hermanos Ameijeiras; durante el período 2008-2018. Resultados: En la pesquisa familiar fueron identificados 177 (15,66 por ciento) nuevos casos de hipercolesterolemia familiar, de ellos se clasifican como casos positivos 35 (19,77 por ciento), casos probables 58 (32,77 por ciento) y casos posibles 84 (47,46 por ciento). Las categorías del estrato Make early diagnosis to prevent early death MEDPED y la edad del caso índice resultaron ser las variables clínicas de interés con mayor probabilidad para identificar nuevos casos de hipercolesterolemia familiar. Conclusiones: los criterios clínicos estandarizados de la escala make early diagnosis to prevent early death P y la edad del caso índice resultaron ser indicadores predictivos de gran valor para identificar y estratificar casos con variantes fenotípicas de hipercolesterolemia familiar(AU)

Introduction: Familial hypercholesterolemia is a disease with high prevalence; it shortens life expectancy if it is not treated, so early diagnosis is essential. Genetic tests are the gold standard for the diagnosis of familial hypercholesterolemia, however, the unavailability of the genetic test should not be an obstacle to proper conduct in these cases. Objective: To identify predictive clinical criteria in the diagnosis by screening of familial hypercholesterolemia. Methods: A prospective descriptive study was carried out from a sample of 393 patients (index cases) of FH at Hermanos Ameijeiras Surgical Clinical Hospital from 2008 to 2018. Results: In the family investigation, 177 (15.66 percent) new cases of familial hypercholesterolemia were identified, 35 of them (19.77 percent) are classified as positive cases, 58 (32.77 percent) as probable cases and 84 as possible cases (47.46 percent)The stratum categories of Make Early Diagnosis to Prevent Early Death (MEDPED) and the age of the index case turned out to be the clinical variables of interest with the greatest probability to identify new cases of familial hypercholesterolemia. Conclusions: The standardized clinical criteria of the make early diagnosis to prevent early death P scale and the age of the index case turned out to be highly valuable predictive indicators to identify and stratify cases with phenotypic variants of familial hypercholesterolemia(AU)

Hipercolesterolemia familiar descripción de un caso en una familia de Armenia - Quindío (Colombia)

Introducción: La Hipercolesterolemia familiar (HF) es una enfermedad genética de carácter autosómico dominante, poco frecuente, generada por la mutación en el cromosoma 19. Es la primera causa de enfermedad cardiovascular prematura. Las mutaciones patogénicas que generan la HF se relacionan con el receptor de LDL (LDLr), la apolipoproteina B-100 (Apo- B100) y la proteína convertasa subtilisina / kexina tipo 9 (PCSK9), que produce elevación del colesterol y alteración de la vía del LDLr en el 80 % de los casos diagnosticados de HF (5). Presentamos un reporte de caso de cuatro pacientes que pertenecen a la misma familia, quienes presentan mutaciones patogénicas de diferente compromiso a nivel cardiovascular y sistémico que ha afectado de manera negativa su cotidianidad. El objetivo de este trabajo es realizar una correlación del hipercolesterolemia familiar de tipo genético a partir de la literatura, con respecto a la serie de casos presentada, y evaluar el impacto que este genera en los servicios de salud, en la vida del paciente y su familia. Discusión: El reporte de caso que presentamos se fundamenta en la sospecha de HF según los criterios de Holanda. En estos pacientes se reconoce mutación del gen LDLr que se relaciona con HF. Sin embargo, no ha sido ampliamente estudiada. Chmara realizó en Polonia por primera vez un estudio en el que reportó la variante ac 11G>T. En Colombia, el estudio de López encontró tres mutaciones, identificadas como variante a c.11G > A, n c.416A > G y c.1187G > A (8). Conclusión: La HF en nuestro medio es poco frecuente y con gran impacto social, en la mayoría de los casos genera síntomas clínicos y aumento del riesgo cardiovascular desde una edad temprana. Es importante resaltar el diagnóstico oportuno y el conocimiento por parte del personal de salud para generar una calidad de vida adecuada a los pacientes y evitar que aumente el riesgo cardiovascular.

Introduction: Familial hypercholesterolemia (FH) is a rare autosomal dominant genetic disease caused by a chromosome 19 mutation. It is the main cause of premature cardiovascular disease. Pathogenic mutations which cause FH are related to the LDL receptor (LDLr), B-100 apolipoprotein (Apo-B100) and type 9 subtilisin/kexin convertase protein (PCSK9), causing blood cholesterol increase and impairment of the LDLr pathway in up to 80% of patients diagnosed with FH. We present the case of 4 patients belonging to the same family and who present pathogenic mutations leading to diverse kinds of cardiovascular and systemic disease. Discussion: The case report we are presenting is based on the suspicion of FH according to the dutch criteria. These patients had the LDLr gene mutation related to FH. However, this mutation has not been thoroughly studied. The ac 11G>T variant was reported for the first time in Poland by Chmara. In Colombia, Lopez found 3 mutations identified as variant a c.11G > A, variant n c.416A > G and variant c.1187G > A. Conclusion: FH is rare in Colombia. Early diagnosis and healthcare worker awareness must be highlighted to improve the quality of life and decrease the cardiovascular risk of patients.

Rastreamento para Hipercolesterolemia Familiar em Pequenos Municípios: A Experiência do Programa HipercolBrasil em 11 Municípios Brasileiros / Screening for Familial Hypercholesterolemia in Small Towns: Experience from 11 Brazilian Towns in the Hipercolbrasil Program

Resumo Fundamento A hipercolesterolemia familiar (HF) é uma doença genética dominante que se caracteriza por níveis sanguíneos elevados de colesterol de lipoproteína de baixa densidade (LDL-C), e está associada à ocorrência de doença cardiovascular precoce. No Brasil, o HipercolBrasil, que é atualmente o maior programa de rastreamento em cascata para HF, já identificou mais de 2.000 indivíduos com variantes genéticas causadoras de HF. A abordagem padrão baseia-se no rastreamento em cascata de casos índices referidos, indivíduos com hipercolesterolemia e suspeita clínica de HF. Objetivos Realizar rastreamento direcionado de 11 pequenos municípios brasileiros com suspeita de alta prevalência de indivíduos com HF. Métodos A seleção dos municípios ocorreu de 3 maneiras: 1) municípios em que houve suspeita de efeito fundador (4 municípios); 2) municípios em uma região com altas taxas de infarto do miocárdio precoce, conforme descrito pelo banco de dados do Sistema Único de Saúde (2 municípios); e 3) municípios geograficamente próximos a outros municípios com alta prevalência de indivíduos com HF (5 municípios). A significância estatística foi considerada como valor p < 0,05. Resultados Foram incluídos 105 casos índices e 409 familiares de primeiro grau. O rendimento dessa abordagem foi de 4,67 familiares por caso índice, o qual é significativamente melhor (p < 0,0001) do que a taxa geral do HipercolBrasil (1,59). Identificamos 36 CIs com variante patogênica ou provavelmente patogênica para HF e 240 familiares de primeiro grau afetados. Conclusão: Nossos dados sugerem que, uma vez detectadas, regiões geográficas específicas justificam uma abordagem direcionada para a identificação de aglomerações de indivíduos com HF.

Abstract Background Familial hypercholesterolemia (FH) is a genetic disease characterized by elevated serum levels of low-density lipoprotein cholesterol (LDL-C), and it is associated with the occurrence of early cardiovascular disease. In Brazil, HipercolBrasil, which is currently the largest FH cascade screening program, has already identified more than 2000 individuals with causal genetic variants for FH. The standard approach is based on cascade screening of referred index cases, individuals with hypercholesterolemia and clinical suspicion of FH. Objectives To perform targeted screening of 11 small Brazilian cities with a suspected high prevalence of people with FH. Methods The selection of cities occurred in 3 ways: 1) cities in which a founder effect was suspected (4 cities); 2) cities in a region with high rates of early myocardial infarction as described by the National Health System database (2 cities); and 3) cities that are geographically close to other cities with a high prevalence of individuals with FH (5 cities). Statistical significance was considered as p value < 0.05. Results One hundred and five index cases and 409 first-degree relatives were enrolled. The yield of such approach of 4.67 relatives per index case was significantly better (p < 0.0001) than the general HipercolBrasil rate (1.59). We identified 36 IC with a pathogenic or likely pathogenic variant for FH and 240 affected first-degree relatives. Conclusion Our data suggest that, once detected, specific geographical regions warrant a target approach for identification of clusters of individuals with FH.

Shortcomings on genetic testing of Familial hypercholesterolemia (FH) in India: Can we collaborate to establish Indian FH registry?

Familial hypercholesterolemia (FH) is a common autosomal dominant disorder that affects ∼1 in 250–500 individuals globally. The only prevalence study in India shows FH in 15% of patients with premature CAD in North Indians. There are only 6 genetic studies in India of the total mutations, 32% are LDLR mutations, 4% are ApoB, 2% are PCSK9 mutations and the mutational spectrum for 37% is unknown. This calls for widespread genetic screening which could help identify definite FH patients. European Atherosclerosis Society-Familial Hypercholesterolemia Studies Collaboration (EAS- FHSC) has taken an initiative to develop a worldwide registry of FH. India is also a part of the collaboration and 3 groups from Mumbai, Delhi and Chennai are actively contributing to this registry. We believe this review might help to understand the Indian scenario of FH and investigators across India can contribute in managing FH in India and further help in the detection, diagnosis and treatment.

Xantomas tuberosos en un paciente pediátrico: la clave en el diagnóstico precoz de una patología grave y poco frecuente / Tuberous xanthomas in a pediatric patient: a clue to the early diagnosis of a severe and infrequent disease

Los xantomas cutáneos reflejan el depósito de lípidos en la piel y pueden ser la única manifestación temprana de dislipidemias de inicio en la infancia. Las características y distribución de los xantomas orientan a la patología de base; los xantomas tuberosos tienen una fuerte asociación con la hipercolesterolemia homocigota familiar, una patología muy infrecuente. Su detección temprana otorga una ventana terapéutica para prevenir la ateroesclerosis acelerada y la mortalidad. Se presenta el caso de una paciente que comenzó a los dos años con xantomas tuberosos, que fueron la clave diagnóstica para identificar la hipercolesterolemia homocigota familiar subyacente.

Cutaneous xanthomas reflect lipid deposition on the skin and may be the only early manifestation of a childhoodonset dyslipidemia. Characteristics and distribution of the xanthomas signalize the underlying pathology, tuberousxanthomas being strongly associated with homozygous familial hypercholesterolaemia, an extremely rare condition. Its early detection provides a therapeutic window to prevent accelerated atherosclerosis and mortality. We present the case of a patient who started at two years with tuberous xanthomas, which were the diagnostic clue to identify the underlying homozygous familial hypercholesterolaemia.

Avaliação do perfil de microRNAs exossomais em portadores de hipercolesterolemia familial / Evaluation of the profile of exosomal microRNAs in patients with familial hypercholesterolemia

A Hipercolesterolemia Familial (HF) é uma doença hereditária do metabolismo lipídico que causa aos portadores alta incidência de aterosclerose prematura. A HF pode ser diagnosticada clínica e geneticamente, entretanto, apenas cerca de 40% podem ter confirmados pelo diagnostico molecular. Assim, outros sistemas de diagnóstico devem ser avaliados. Ultimamente devido a estabilidade em fluidos biológicos, os exossomos circulantes apresentam grande potencial, pois carreiam um número variado de compostos e são considerados veículos de intercomunicação entre os tecidos. Sabe-se que vários RNAs são carreados nos exossomos, incluindo miRNAs, lncRNA e uma variedade de proteínas. Estes componentes podem ser marcadores de diagnóstico para várias doenças inclusive a HF e suas complicações cardiovasculares. Foram utilizadas amostras de exossomos plasmáticos provenientes de 54 pacientes HF sem uso de estatina por, no mínimo, seis semanas, e 38 indivíduos normolipidêmicos para sequenciamento de miRNAs e estudo da proteômica. Os exossomos foram isolados utilizando dois métodos precipitação química e cromatográfica de exclusão de tamanho e caraterizados utilizando: dispersão de luz dinâmica, Western blotting, rastreamento de nanopartículas (NanoSight), imunomarcação e microscopia eletrônica de transmissão. Os miRNAs e proteínas foram extraídos dos exossomos e analisados por sequenciamento de nova geração e espectrometria de massa, respectivamente. Os dados clínicos, biodemográficos e laboratoriais dos pacientes HF e controles indicaram diferenças significativas esperadas entre os grupos, indicando que foram selecionados adequadamente. A caracterização físico-química dos exossomos mostrou resultados com tamanho de ˜90nm e imunorreação positiva para tetraspaninas. O resultado do sequenciamento identificou acima 2000 miRNAs. Os miR-122- 5p e miR-21-5p apresentaram expressão aumentada no grupo HF (log2FC=1,79 e log2FC=1,27, respectivamente), e o miR-122-5p pós normalização em relação ao controle manteve significativo comparados ao controle (p=0,034). A análise comparativa entre exossomos e plasma total mostrou diferença significativa, pois foram identificadas 239 proteínas (p <0,05) diferentes entre exossomos e plasma. Em exossomos, 17 proteínas foram aumentadas e 21 diminuídas em pacientes com HF em comparação com o controle (p <0,05). Destas, seis proteínas foram mais abundantes em HF e sete proteínas foram menos abundantes em exossomos de pacientes com HF em comparação com o controle. A análise de enriquecimento por bioinformática mostrou que a maior parte dessas moléculas (miRNAs e proteínas) foram relacionadas com metabolismo lipídico, dislipidemia, aterosclerose, doença arterial coronariana, adipogênese. Assim, na busca de novos alvos como potenciais biomarcadores de diagnóstico da HF, nossos resultados da análise integrativa entre os miRNAs e as proteínas exossomais abre novas frentes de pesquisa mais bem direcionadas, para a validação desses miRNAs e proteínas exossomais

Familial Hypercholesterolemia (FH) is an inherited disease of lipid metabolism that causes a high incidence of premature atherosclerosis in patients. FH can be diagnosed clinically and genetically, however, only about 40% can be confirmed by molecular diagnosis. Thus, other diagnostic systems should be evaluated. Lately, due to stability in biological fluids, circulating exosomes have great potential, as they carry a varied number of compounds and are considered vehicles of intercommunication between tissues. Several RNAs are known to be carried on exosomes, including miRNAs, lncRNA, and a variety of proteins. These components can be diagnostic markers for several diseases including FH and its cardiovascular complications. Plasma exosome samples from 54 FH patients without statin use for at least six weeks and 38 normolipidemic individuals were used for miRNA sequencing and proteomics studies. Exosomes were isolated using two methods chemical precipitation and size exclusion chromatography and characterized using: dynamic light scattering, Western blotting, nanoparticle tracking (NanoSight), immunostaining and transmission electron microscopy. MiRNAs and proteins were extracted from exosomes and analyzed by next-generation sequencing and mass spectrometry, respectively. Clinical, biodemographic and laboratory data of FH patients and controls indicated significant expected differences between the groups, indicating that they were appropriately selected. The physicochemical characterization of exosomes showed results with a size of ˜90nm and positive immunoreaction for tetraspanins. The sequencing result identified above 2000 miRNAs. miR-122-5p and miR-21-5p showed increased expression in the FH group (log2FC=1.79 and log2FC=1.27, respectively), and miR122-5p after normalization in relation to the control remained significant compared to the control (p=0.034). The comparative analysis between exosomes and total plasma showed a significant difference, as 239 different proteins (p < 0.05) were identified between exosome and plasma. In exosomes, 17 proteins were increased and 21 decreased in FH patients compared to control (p < 0.05). Of these, six proteins were more abundant in FH and seven proteins were less abundant in exosomes from patients with FH compared to the control. Bioinformatics enrichment analysis showed that most of these molecules (miRNAs and proteins) were related to lipid metabolism, dyslipidemia, atherosclerosis, coronary artery disease, adipogenesis. Thus, in the search for new targets as potential diagnostic biomarkers of FH, our results of the integrative analysis between miRNAs and exosomal proteins opens new and better-directed research fronts for the validation of these miRNAs and exosomal proteins

Avaliação do perfil de metilação dos genes LDLR, PCSK9, LDLRAP1 em pacientes portadores de hipercolesterolemia familial / Evaluation of the methylation profile of LDLR, PCSK9, LDLRAP1 genes in patients with familial hypercholesterolemia

As variabilidades genotípicas que determinam algumas alterações fenotípicas e metabólicas podem ter seu diagnostico falho se baseado apenas nos dados genômicos. Na hipercolesterolemia familial (HF) pode-se observar que os dados de variantes nos genes da LDLR, PCSK9, APOB e LDLRAP1 sugeridos pelos consensos atuais para confirmar o diagnóstico, tem mostrado serem insuficientes, mostrando baixa porcentagem de confirmação, mesmo nos dos casos em que características fenotípicas apresentam dados sugestivos importantes. A complementação no auxílio diagnóstico com dados epigenéticos tem sido sugerida em muitas doenças, principalmente nas crônicos degenerativos. A metilação do DNA pode estar envolvida no mecanismo que regulam vários processos metabólicos, entre os quais os envolvidos na expressão de proteínas e neste estudo os que estão envolvidos no metabolismo do colesterol, que poderia explicar fenótipos hipercolesterolemicos sem demonstração clara de variantes nos genes de consenso. O objetivo do presente estudo foi comparar o perfil de metilação dos genes LDLR, PCSK9 e LDLRAP1 entre pacientes com diagnóstico de Hipercolesterolemia Familial confirmado através de variantes genéticas descritas na literatura e pacientes sem diagnóstico confirmado. Além da comparação com indíviduos normolipidêmicos. A seleção dos indivíduos foi realizada na Seção de Dislipidemia do Instituto Dante Pazzanese de Cardiologia (IDPC), do Departamento de Análises Clínicas e Toxicológicas da Universidade Federal do Rio Grande do Norte (UFRN), da Universidade Estadual de Campinas (UNICAMP) e da Faculdade de Medicina de São José do Rio Preto (FAMERP). Através dos critérios MEDPED foram selecionados 133 pacientes para a realização do sequenciamento de um painel de genes relacionados ao fenótipo de HF e a homeostasia do colesterol a fim de confirmar o diagnóstico. Todos os pacientes tiveram o DNA purificado, que foi submetido ao tratamento com bissulfito, amplificado, purificado, desnaturado e sequenciado no sistema PyroMark Q24. Avaliou-se o perfil de metilação, em sítio CpG dos genes da LDLR, PCSK9 e LDLR AP1. A análise estatística foi realizada utilizando o software SPSS v.19, GraphPad Prism, versão 1.03 e o e o software R. 4.1.0. Os pacientes foram classificados em Grupo I: Pacientes com diagnóstico molecular confirmado pelo estudo fenotípico e genotipico (n=40); Grupo II: Pacientes fenotipicamente determinados como hipercolesterolemico, mas sem diagnóstico molecular confirmado pelo estudo genomico (n=93); Grupo III: indivíduos fenotipicamente determinados normolipidêmicos de acordo com a V Diretriz Brasileira de Dislipidemia (n=23). A análise comparativa entre os grupos I x II e II x III, demonstrou diferença estatísta significativa em 13 sítios CpG do total de 28 sítios CpG analisados nos três genes. Além disso, foi possível concluir que alterações nos sítios CpG presentes no gene LDLR influenciaram na presença de xantomas e arco córneo. Houve correlação positiva entre a idade e perfil de metilação do gene PCSK9, assim como, alterações nos sítios CpG deste gene influenciaram na presença de arco córneo e IAM. Além disso, alterações no sítio CPG presente no gene LDLRAP1 influenciou no desenvolvimento de DAC, IAM e RM, além da presença de xantelasma

The genotypic variabilities that determine some phenotypic and metabolic alterations can be misdiagnosed if based only on genomic data. In familial hypercholesterolemia (FH) it can be observed that the data of variants in the genes of LDLR, PCSK9, APOB and LDLR AP1 suggested by the current consensus to confirm the diagnosis, has shown to be insufficient, showing a low percentage of confirmation, even in the cases in which phenotypic characteristics present important suggestive data. Complementing the diagnostic aid with epigenetic data has been suggested in many diseases, especially in chronic degenerative diseases. DNA methylation may be involved in the mechanisms that regulate several metabolic processes, including those involved in the expression of proteins that ,in this study, are involved in cholesterol metabolism, which could explain hypercholesterolemic phenotypes without a clear demonstration of variants in consensus genes. The aim of the present study was to compare the methylation profile of LDLR, PCSK9 and LDLRAP1 genes between patients with a diagnosis of Familial Hypercholesterolemia confirmed through genetic variants described in the literature and patients without a confirmed diagnosis. In addition to the comparison with normolipidemic individuals. The selection of individuals was carried out at the Dyslipidemia Section of the Instituto Dante Pazzanese de Cardiologia (IDPC), in the Department of Clinical and Toxicological Analyzes of the Federal University of Rio Grande do Norte (UFRN), in the State University of Campinas (UNICAMP) and in the Faculdade of Medicine of São José do Rio Preto (FAMERP). Through the MEDPED criteria, 133 patients were selected to perform the sequencing of a panel of genes related to the FH phenotype and cholesterol homeostasis in order to confirm the diagnosis. All patients had their DNA purified, which were subjected to bisulfite treatment, amplified, purified, denatured and sequenced on the PyroMark Q24 system. The methylation profile in the CpG site of the LDLR, PCSK9 and LDLRAP1 genes were evaluated. Statistical analysis were performed using SPSS v.19 software, GraphPad Prism, version 1.03 and R. 4.1.0 software. Patients were classified into Group I: Patients with a molecular diagnosis confirmed by phenotypic and genotypic studies (n=40); Group II: Patients phenotypically determined to be hypercholesterolemic, but without a molecular diagnosis confirmed by the genomic study (n=93); Group III: phenotypically determined normolipidemic individuals according to the V Brazilian Dyslipidemia Directive (n=23). The comparative analysis between groups I x II and II x III showed a statistically significant difference in 13 CpG sites of the total of 28 CpG sites analyzed in the three genes of the project. Furthermore, it was possible to conclude that alterations in the CpG sites present in the LDLR gene influenced the presence of xanthomas and arc corneum. There was a positive correlation between age and PCSK9 gene methylation profile, as well as changes in the CpG sites of this gene influenced the presence of arc corneum and AMI. In addition, alterations in the site present in the LDLRAP1 gene are influencing the development of CAD, AMI and MR, in addition to the presence of xanthelasma

Clinical and molecular characteristics of a family with familial hypercholesterolemia with LDLRAP1 and ABCG8 gene abnormality / 中华检验医学杂志

Objective:To study the clinical and molecular characteristics of a family with familial hypercholesterolemia (FH) with LDLRAP1 and ABCG8 gene abnormality.Methods:In September 2020, one case of FH was included in Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine; peripheral venous blood samples of members of the family were collected to detect serum total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) indicators; use high-performance liquid chromatography to detect serum stigmasterol and sitosterol content; perform second-generation gene sequencing to detect gene mutations in probands and family members; use Pymol software to detect gene mutations point for pathogenicity analysis, and use Uniprot Modelling software to perform protein structure modeling.Results:The patient presented with anemia, multiple xanthomas and early-onset acute coronary syndrome. The coronary angiography showed severe coronary artery lesions; abdominal ultrasound showed splenomegaly; blood smear showed shaped erythrocytes and large platelets. The level of serum TC, LDL-C, stigmasterol and sitosterol was 8.54 mmol/L (2.3-5.7 mmol/L), 4.84 mmol/L (range of normal value 1.3-4.3 mmol/L), 44 μmol/L (1.0-10 μmol/L), 28 μmol/L (1.0-15 μmol/L), respectively; LDLRAP1 gene mutation was found: exon4 c.415C>T:p.Q139X; the truncated protein formed by this homozygous mutation lost multiple stable protein structure regions, which can not have a normal function. At the same time, ABCG8 gene mutations were also found: exon13 c.1895T>C (p.V632A) and exon8 c.1199C>A:p.T400K . Two cases of family members had a mild increase in HDL-C (Ⅱ5: 2.33 mmol/L, Ⅱ6∶2.96 mmol/L), 3 cases carrying the ABCG8 gene mutations had a slight increase in stigmasterol (Ⅱ8: 23 μmol/L, Ⅱ7: 24 μmol/L, Ⅰ2: 18 μmol/L) and sitosterol (Ⅱ8: 41 μmol/L, Ⅱ7: 33 μmol/L, Ⅰ2: 45 μmol/L), suggesting that its association with the concentration of plant sterols. Conclusions:FH patients with LDLRAP1 and ABCG8 gene abnormalities may have abnormal plant sterol concentrations, and their clinical manifestations are more complicated. Therefore, family history, LDL-C, plant sterol levels, and genetic test results should be considered comprehensively.

Portador Heterozigoto Composto de Hipercolesterolemia Familiar Causada por Variantes no LDLR / Compound Heterozygous Familial Hypercholesterolemia Caused by LDLR Variants

Resumo A hipercolesterolemia familiar (HF) é uma doença genética causada por um defeito primário no gene que codifica o receptor da LDL. Mutações diferentes no mesmo gene caracterizam um heterozigoto composto, mas pouco se sabe sobre o fenótipo dos portadores. Portanto, neste estudo, descrevemos o rastreamento em cascata de uma família brasileira com essa característica. O caso-índice é um homem de 36 anos, com colesterol total (CT) de 360 mg/dL (9,3 mmol/L) e concentração de LDL-c de 259 mg/dL (6,7 mmol/L), além de xantomas de tendão de Aquiles, obesidade e pré-hipertensão. A genotipagem identificou as mutações 661G>A, 670G>A e 682G>A, no exon 4, e 919G>A, no exon 6. A mesma mutação no exon 4 foi observada no filho do caso-índice (7 anos), que também tem hipercolesterolemia e xantomas tendinosos, ao passo que a filha do caso-índice (9 anos) apresenta mutação no exon 6 e hiperlipidemia, sem xantomas. Em suma, este relato permite uma melhor compreensão acerca da base molecular da HF no Brasil, um país multirracial, onde é esperada uma população heterogênea.

Abstract Familial hypercholesterolemia (FH) is a genetic disease caused by a primary defect in the LDL-receptor gene. Distinct variants in the same gene characterize a compound heterozygote, but little is known about the phenotypes of the carriers. Therefore, herein, we describe the cascade screening of a Brazilian family with this characteristic. The index case, a 36-year-old male, had a total cholesterol level of 360 mg/dL (9.3 mmol/L) and LDL-c value of 259 mg/dL (6.7 mmol/L), in addition to Achilles tendon xanthomas, obesity and prehypertension. Genotyping identified the variants 661G>A, 670G>A, 682G>A in exon 4 and 919G>A in exon 6. The same variant in exon 4 was found in the index case's son (7-y), who also had hypercholesterolemia and xanthomas, while the index case's daughter (9-y) had the variant in exon 6 and hyperlipidemia, without xanthomas. In summary, this report allows for a better insight into the molecular basis of FH in Brazil, a multi-racial country where a heterogeneous population is expected.

Phenotype of definite familial hypercholesterolemia with negative genetic study in Argentina / Fenotipo de hipercolesterolemia familiar definitivo con estudio genético negativo en Argentina

Abstract Objective: Familial hypercholesterolemia (FH) is a monogenic disease, associated with variants in the LDLR, APOB and PCSK9 genes. The initial diagnosis is based on clinical criteria like the DLCN criteria. A score > 8 points qualifies the patient as "definite" for FH diagnosis. The detection of the presence of a variant in these genes allows carrying out familial cascade screening and better characterizes the patient in terms of prognosis and treatment. Methods: In the context of the FH detection program in Argentina (Da Vinci Study) 246 hypercholesterolemic patients were evaluated, 21 with DLCN score > 8 (definite diagnosis).These patients were studied with next generation sequencing to detect genetic variants, with an extended panel of 23 genes; also they were adding the large rearrangements analysis and a polygenic score of 10 SNP (single nucleotide polymorphism) related to the increase in LDL-c. Results: Of the 21 patients, 10 had variants in LDLR, 1 in APOB with APOE, 1 in LIPC plus elevated polygenic score, and 2 patients showed one deletion and one duplication in LDLR, the later with a variation in LIPA. It is highlighted that 6 of the 21 patients with a score > 8 did not show any genetic alteration. Conclusions: We can conclude that 28% of the patients with definite clinical diagnosis of FH did not show genetic alteration. The possible explanations for this result would be the presence of mutations in new genes, confusing effects of the environment over the genes, the gene-gene interactions, and finally the impossibility of detecting variants with the current available methods.

Resumen Objetivo: La hipercolesterolemia familiar (HF) es una enfermedad monogénica asociada a variantes en los genes RLDL, APOB y PCSK9. El diagnóstico inicial se basa en criterios clínicos, como el de la red de clínica de lípidos holandesa (DLCN). Un puntaje > 8 puntos califica al paciente como "definitivo" para diagnóstico de HF. La identificación de una variante en estos genes permite realizar el cribado en cascada familiar y caracterizar mejor al paciente en cuanto al pronóstico y el tratamiento. Métodos: En el marco del Programa de Detección de HF en Argentina (Estudio Da Vinci) se evaluó a 246 pacientes hipercolesterolémicos, 21 con puntaje DLCN > 8 (diagnóstico definitivo). Se estudió a estos pacientes con secuenciación de próxima generación para reconocer variantes genéticas, con un panel ampliado de 23 genes, sumado al análisis de grandes rearreglos y por último se aplicó un score poligénico de 10 SNP (polimorfismo de nucleótido único) relacionados con aumento del c-LDL. Resultados: De los 21 pacientes, 10 presentaron variantes en RLDL, uno en APOB junto a APOE, uno en LIPC más puntaje poligénico elevado, dos pacientes con una deleción y una duplicación en RLDL y este último caso con una variante en LIPA. Es destacable que 6 de los 21 pacientes con puntaje DLCN > 8 no mostraron ninguna alteración genética. Conclusiones: El 28% de los pacientes con diagnóstico clínico definitivo de HF no evidenció alteración genética. Las posibles explicaciones de este resultado serían la presencia de mutaciones en nuevos genes, los efectos confundidores del ambiente sobre los genes o la interacción gen-gen y por último la imposibilidad de detectar variantes con la metodología actual disponible.

Minimizing the risk of perioperative cardiovascular complications in homozygous familial hypercholesterolemia: a case report

Homozygous familial hypercholesterolemia (HoFH) is a rare inherited disorder that presents as abnormally elevated levels of low-density lipoprotein cholesterol and premature heart disease, requiring frequent intervention through lipid apheresis for management. The risk of perioperative cardiac events is higher in patients with HoFH because of its pathophysiological manifestations in the vascular system. Careful cardiac precautions and anesthetic assessments are necessary to ensure patient safety. In the following case report, we discuss the clinical course and anesthetic considerations for a 14-year-old girl with HoFH undergoing sedation for dental extractions and mandibular molar uprighting in an outpatient oral surgery clinic. Considerations included the use of heparin in the patient's weekly plasma lipid apheresis treatment. In order to reduce the risks of peri- and postoperative bleeding and perioperative cardiac events, the operation was scheduled for 4 days after apheresis. This allowed for adequate heparin clearance, while also reducing the likelihood of possible cardiac events. A literature review revealed no results for the outpatient management of patients with HoFH undergoing sedation for noncardiac procedures. Our reported case serves as a clinical example for physicians to be utilized in the future.

Hipercolesterolemia familiar. Enfoque desde la Pediatría / Familial hypercholesterolemia. A pediatric focus

La Hipercolesterolemia Familiar (HF) es una enfermedad hereditaria frecuente que se caracteriza por niveles elevados de colesterol ligado a las lipoproteínas de baja densidad (C-LDL). El exceso de LDL se acumula en las arterias produciendo aterosclerosis prematura. El diagnóstico y tratamiento desde la infancia mejoran el pronóstico de la enfermedad. Existe subdiagnóstico de la HF lo que provoca muertes prematuras por enfermedad cardiovascular (ECV). Para mejorar el subdiagnóstico la Sociedad Argentina de Pediatría propuso en el año 2015 realizar tamizaje universal al ingreso escolar. Es relevante entonces que el pediatra pueda diagnosticar la hipercolesterolemia y diferenciar las hipercolesterolemias monogénicas o familiares, de las secundarias (AU)

Familial hypercholesterolemia (FH) is a common hereditary disease that is characterized by high cholesterol levels, linked to low-density lipoproteins (LDL). Excess LDL accumulates in the arteries leading to premature atherosclerosis. Early diagnosis and treatment since childhood improve the prognosis of the disease. FH is underdiagnosed resulting in premature death due to cardiovascular disease (CVD). To improve diagnosis, in 2015 the Argentine Society of Pediatrics proposed a universal screening program at school age. It is relevant, therefore, for the pediatrician to be able to diagnose hypercholesterolemia and differentiate monogenic or familial from secondary hypercholesterolemia (AU)

Prevalence of familial hypercholesterolemia in premature coronary artery disease patients admitted to a tertiary care hospital in North India

Aims The prevalence of premature coronary artery disease (CAD) in India is two to three times more than other ethnic groups. Untreated heterozygous familial hypercholesterolemia (FH) is one of the important causes for premature CAD. As the age advances, these patients without treatment have 100 times increased risk of cardiovascular (CV) mortality resulting from myocardial infarction (MI). Recent evidence suggests that one in 250 individuals may be affected by FH (nearly 40 million people globally). It is indicated that the true global prevalence of FH is underestimated. The true prevalence of FH in India remains unknown. Methods A total of 635 patients with premature CAD were assessed for FH using the Dutch Lipid Clinical Network (DLCN) criteria. Based on scores, patients were diagnosed as definite, probable, possible, or no FH. Other CV risk factors known to cause CAD such as smoking, diabetes mellitus, and hypertension were also recorded. Results Of total 635 patients, 25 (4%) were diagnosed as definite, 70 (11%) as probable, 238 (37%) as possible, and 302 (48%) without FH, suggesting the prevalence of potential (definite + probable) FH of about 15% in the North Indian population. FH is more common in younger patients, and they have lesser incidence of common CV risk factors such as diabetes, hypertension, and smoking than the younger MI patients without FH (26.32% vs.42.59%; 17.89% vs.29.44%; 22.11% vs.40.74%). Conclusion FH prevalence is high among patients with premature CAD admitted to a cardiac unit. To detect patients with FH, routine screening with simple criteria such as family history of premature CAD combined with hypercholesterolemia, and a DLCN criteria score >5 may be effectively used.

Characteristics of Acute Cerebral Infarction in Patients with Familial Hypercholesterolemia

BACKGROUND: The patients with familial hypercholesterolemia (FH) suffer from early onset atherosclerotic vascular disease due to high level of cholesterol and subsequent vascular inflammation, especially in the form of coronary artery disease. We investigated the clinical characteristics of FH associated cerebral infarction and its possible mechanism. METHODS: Between January 2014 and May 2017, acute cerebral infarction patients who admitted to Chung-Ang University Hospital were reviewed from stroke registry and the diagnosis of FH was made based on the Dutch Lipid Clinic Network Diagnostic Criteria for FH. We reviewed their initial laboratory and brain imaging information, prescribed medication and followed lipid profile after discharge. Stroke mechanism was determined based on Trial of ORG 10172 in Acute Stroke Treatment classification. RESULTS: Among 1,401 acute cerebral infarction or transient ischemic attack patients, one probable and three possible FH stroke patients were detected. All the patients denied of previous coronary artery disease history and initial lipid panel revealed high levels of total cholesterol (378±75 mg/dL) and low-density lipoprotein-cholesterol (238±56 mg/dL). Stroke mechanisms were heterogeneous, including one atherosclerotic, two vertebral artery dissection cases and one coagulation disorder. All the patients were combined with noticeable degree of intracranial atherosclerosis and were maintained with statin treatment. CONCLUSIONS: This study illustrates diverse stroke mechanism among stroke patients with FH. Further research is required to disclose exact incidence of FH among stroke population and effective treatment strategy.

Caracterização funcional in vitro de variantes no gene PCSK9 identificadas em pacientes com Hipercolesterolemia Familial / In vitro functional characterization of PCSK9 variants identified in patients with Familial Hypercholesterolemia

A Hipercolesterolemia Familial (HF) é uma doença genética do metabolismo das lipoproteínas, caracterizada pelo aumento do colesterol plasmático, transportado principalmente pela lipoproteína de baixa densidade (LDL). A HF é causada principalmente por mutações nos genes LDLR, APOB e PCSK9. As mutações conhecidas na PCSK9 podem levar ao aumento ou diminuição da função proteolítica da proteína, as quais são associadas ao aumento ou diminuição da LDL-c plasmática, respectivamente. Com o projeto genoma humano surgiram novos métodos de sequenciamento, o que resultou em um grande número de novas variantes genéticas relacionadas à HF. Entretanto, os mecanismos pelos quais essas variantes influenciam na concentração do colesterol e sua interferência na resposta terapêutica não estão totalmente elucidados. O objetivo do presente trabalho foi avaliar in vitro o efeito de variantes na região codificadora e reguladora do gene PCSK9 identificadas em pacientes HF utilizando sequenciamento de nova geração. Para a caracterização funcional das variantes na região codificadora da PCSK9, primeiramente foi avaliado o impacto dessas variantes na interação PCSK9-LDLR via Docking molecular. Células HEK293FT foram transfectadas com as diferentes construções da PCSK9, e posteriormente, foram utilizadas em ensaios para avaliar a atividade do LDLR e a internalização de LDL por citometria de fluxo. Para as variantes na região reguladora da PCSK9, foi realizado uma predição in silico do possível efeito de variantes na região 3UTR na ligação de miRNAs. A avalição da interação entre os miRNAs preditos, e a região 3UTR da PCSK9, e o possível impacto nessa interação na presença de variantes na região 3UTR, foi realizada em células HEK293FT transfectadas com um plasmídeo contendo a 3UTR da PCSK9 e um gene repórter da Gaussia luciferase, juntamente com um plasmídeo de expressão contendo os miRNAs de interesse. Foi também estudado o efeito dos miRNAs preditos sobre a expressão, RNAm e proteína, da PCSK9 via RT-qPCR e Western blot, em células HepG2. Foram identificadas 9 variantes na região codificadora da PCSK9, e duas, E32K e R469W, foram selecionadas para os ensaios posteriores. Para a R469W foi observada uma possível alteração conformacional a qual poderia aumentar a afinidade da PCSK9 pelo LDLR. Para a E32K, uma possível associação com HF foi observada em uma família brasileira com ascendência japonesa. As variantes E32K e R469W apresentaram uma redução na atividade do LDLR de 5 e 11%, respectivamente em comparação a PCSK9-WT. Entretanto, não foram observadas reduções estaticamente significativas na atividade do LDLR e na internalização da LDL em células transfectadas com ambas as variantes. Dez variantes foram encontradas na região 3UTR da PCSK9, entre elas três foram selecionadas por impactar a ligação de quatro miRNAs. Nossos dados demonstraram uma redução significativa na expressão da PCSK9 em células HepG2 transfectadas com os miR-4721 e miR-564 (p=0,036 e p=0,010, respectivamente). Porém, não foi observada diferenças na expressão da luciferase em células transfectadas com esses miRNAs, não sendo possível validar a interação miRNA-RNAm. As variantes no gene PCSK9 identificadas no nosso estudo podem não explicar individualmente o fenótipo HF, mas podem contribuir para a severidade da doença juntamente com outras variantes em outros genes

Familial Hypercholesterolemia (FH) is a genetic disorder of lipoprotein metabolism, characterized by elevated plasma cholesterol levels, mostly carried by low-density lipoprotein (LDL). FH is mainly caused by mutations in three genes, LDLR, APOB, and PCSK9. Gain-of-function mutations in PCSK9 reduce LDL receptor levels, resulting in high levels of LDL cholesterol in the plasma. Loss-of-function mutations lead to higher levels of the LDL receptor, resulting in lower LDL cholesterol levels. The Human Genome Project led to a faster technological development related to sequencing methods, which allowed identifying many novel variants associated with FH. However, the mechanisms by which these variants influence cholesterol levels and their interference in therapeutic response are not fully understood. The aim of the present study was to perform an in vitro characterization of the effect of PCSK9 variants identified in FH patients using Next-Generation Sequencing. For the functional characterization of variants in the coding region of PCSK9, the impact of these variants on PCSK9-LDLR interaction was evaluated by molecular docking. HEK293FT cells were transiently transfected with different PCSK9 constructs, and the amount of cell surface LDLR and LDL internalization were determined by flow cytometry. For the variants in PCSK9 3UTR region, an in silico prediction of PCSK9 3UTR variants in miRNA seed regions and target sites was performed. To determine whether the predicted miRNAs directly interact with PCSK9 3UTR region, HEK293FT cells were co-transfected with a vector containing a PCSK9 3'UTR region and a Gaussia luciferase reporter gene, together with an expression plasmid containing the miRNAs of interest. The effect of the predicted miRNAs on the expression of PCSK9 was evaluated using RT-qPCR and Western blot in HepG2 cells transiently transfected with miRNA mimics. Nine missense variants were identified in PCSK9 gene. E32K e R469W were chosen for further analysis. For R469W, a possible conformational change was observed that could increase the affinity of PCSK9 for LDLR, when compared to the wild-type. For E32K, a possible association with FH in a Brazilian family with Japanese ancestry was observed. E32K and R469W had a 5% and 11% decreased level of cell surface LDLR, respectively, as compared with WT-PCSK9. However, no significant reduction in the number of cell surface LDLR and LDL internalization was observed in transfected cells for both variants. Ten variants were found in PCSK9 3'UTR region, of which three were selected for affecting the binding of four miRNAs. Our data demonstrated a significant downregulation of PCSK9 in cells transfected with miR-4721 and miR-564 miRNA mimics, compared to cells transfected with a scramble control (p=0,036 and p=0,010, respectively). However, no differences in luciferase expression were observed in cells transfected with these miRNAs, therefore, it was not possible to experimentally validate miRNA-mRNA interaction. PCSK9 variants found in our study may not fully explain FH phenotype but may contribute to the severity of the disease together with other variants in other genes