Determination of sdLDL particles in patients with Familial Hyercholesterolaemia and Familial Combined Hyperlipidaemia

Several studies demonstrated that sdLDL are an emerging cardiovascular (CV) risk factor. The objective of this study was sdLDL measurement in patients with genetic diagnosis of Familial Hypercholesterolaemia (FH) and clinical diagnosis of Familial Combined Hyperlipidaemia (FCHL) to establish a relation between sdLDL, CV risk and the efficacy of therapeutics. Lipid profile was determined using a polyacrylamide gel electrophoresis system that separates the particles in serum that contain cholesterol. The lipidogram obtained classifies the patients as being profile A (low CV risk) or B (high CV risk) depending on the sdLDL concentration. The lipid profile was obtained from 43 FH adults and 46 FCHL adults, index and relatives. FH and FCHL patients without medication and with high sdLDL (>6mg/dl) have significant higher levels of total cholesterol, LDL and ApoB and FCHL patients also have significant higher triglycerides, compared to FH and FCHL patients with sdLDL levels under recommended values. Under medication FH patients have significant higher ApoB levels and lower HDL, and FCHL patients have significant higher total cholesterol, LDL and ApoB levels. Interestingly, 71,4% of FCHL patients under medication presented high CV risk profile, showing that statins seem not to decrease sdLDL levels and neither CV risk. Also FCHL patients are not well medicated or do not respond to usual medication to decrease cholesterol. These preliminary results indicate that sdLDL could be a good biomarker for treatment control but further studies are needed to evaluate the effect of medication in sdLDL levels in FH and FCHL patients.Alexandra Gomes was funded by FCT PIC/IC/83020/2007, Tânia Santos was funded by FCT PIC/IC/83333/2007 and project grant FCT PIC/IC/83333/200

Low-density lipoprotein receptor mutational analysis in diagnosis of familial hypercholesterolemia

PURPOSE OF REVIEW: To present up to date evidence on the pathogenicity of low-density lipoprotein receptor (LDLR) variants and to propose a strategy that is suitable for implementation in the clinical work-up of familial hypercholesterolaemia. RECENT FINDINGS: More than 1800 variants have been described in the LDLR gene of patients with a clinical diagnosis of familial hypercholesterolaemia; however, less than 15% have functional evidence of pathogenicity. SUMMARY: The spectrum of variants in the LDLR identified in patients with clinical familial hypercholesterolaemia is increasing as novel variants are still being reported. However, over 50% of all LDLR variants need further evidence before they can be confirmed as mutations causing disease. Even with applying the recent American College of Medical Genetics variant classification, a large number of variants are still considered variants of unknown significance. Before obtaining an undisputable confirmation of the effect on the expression and activity of the LDLR, reporting these variants as part of a clinical diagnosis to the patient holds the risk that it might need to be withdrawn in a later stage. An investment should be made to develop functional assays to characterize LDLR variants of unknown significance for a better patient diagnosis and to prevent confusion in the physician's office.The MB research has been supported by grants from the Portuguese Cardiology Society, Science and Technology Foundation and BioISI, centre grant UID/MULTI/ 04046/2013, from FCT/MCTES/PIDDAC, Portugal.info:eu-repo/semantics/publishedVersio

A rare polymorphism in the low density lipoprotein (LDL) gene that affects mRNA splicing

Familial hypercholesterolaemia (FH) is usually caused by mutations in the low density lipoprotein (LDL) receptor gene (LDLR) that impair clearance of LDL from the circulation. The increased risk of premature coronary heart disease associated with FH can be reduced by dietary advice and treatment with lipid-lowering drug therapy, but it is important to identify affected individuals at an early stage. Several programmes for genetic diagnosis of FH that rely on identifying nucleotide substitutions in genomic DNA have been initiated, but the validity of these is dependent on distinguishing between a silent nucleotide variant and a mutation that affects LDL-receptor function. Here we describe a single nucleotide substitution in the coding region of exon 9 of LDLR that is an apparently silent polymorphism: CGG (Arg406) to AGG (Arg). Analysis of mRNA from the patient's cells showed that the mutation introduces a new splice site that is used to the exclusion of the natural splice site and causes a deletion of 31 bp from the mRNA, predicted to introduce premature termination four codons after R406. This finding emphasizes the caution needed in genetic diagnosis of FH based on genomic DNA sequence alone

Estudo Português de Hipercolesterolemia Familiar

A Hipercolesterolemia Familiar (FH) é uma doença autossómica dominante que se caracteriza, a nível clínico, por níveis elevados de colesterol LDL, levando ao aparecimento prematuro de doenças cardiovasculares (DCV). A nível genético esta doença caracteriza-se, principalmente, por mutações em três genes: LDLR, APOB e PCSK9. Estima-se que em Portugal existam cerca de 20 000 doentes com FH. A identificação clínica de FH é possível mas apenas o estudo molecular confirma a presença da doença. O Estudo Português de Hipercolesterolemia Familiar (EPFH) tem como objectivo principal identificar a causa genética da dislipidémia em doentes com diagnóstico clínico de FH. O EPHF recebeu desde 1999, para realização do estudo molecular, 486 casos-index com diagnóstico clínico de FH e 858 familiares. O estudo molecular é realizado em 3 fases. Fase I: Identificação de mutações nos genes APOB e LDLR. Fase II: Pesquisa de grandes rearranjos no gene LDLR por MLPA. Fase III: Pesquisa de mutações no gene PCSK9. A pesquisa de mutações nos genes APOB e PCSK9 é realizada por amplificação dos fragmentos a estudar e sequenciação directa. No gene LDLR os 18 exões são amplificados dos 18 exôes por PCR e analisados por DHPLC e sequenciação. Até à data foram identificados um total de 504 doentes com um defeito genético num dos três genes estudados: 3 doentes com mutação no gene PCSK9, 12 doentes com mutação no gene APOB e 438 doentes com mutação no gene LDLR (7 dos quais em homozigotia ou heterozigotia composta). No gene LDLR foram encontradas 89 mutações diferentes, que incluem 43 mutações missense,17 delecções/inserções, 6 nonsense, 12 mutações de splicing, 4 grandes delecções e 2 no promotor e 1no codão stop. As mutações mais comuns na população portuguesa são: p.A431T (11%), p.D224N (6,9%) e p.R406W (6,2%). Foram efectuados funcionais em algumas mutações de splicing e comprovou-se a sua patogeneicidade em 6 alterações (c.-135C>G; c.-190+4insTG; c.313+6T>C; c.818-2A>G; c.2389G>T (V776L); c.2547+1G>A). Foram também efectuados estudos funcionais para 5 alterações missense não descritas anteriormente (p.V429L, p.W490R, p.S648P, p.P685S e p.V859M), verificou-se que apenas a alteração p.V859M não é patogenica. No gene APOB foi identificada a mutação mais comum (p.Arg3527Gln) e também a mutação p.Tyr3560Cys. No gene PCSK9 foi encontrada uma única alteração, p.Asp374His. A FH esta sub-diagnosticada no nosso País, esforços têm de ser conduzidos para identificar estes doentes, ainda em idade jovem, de modo a que seja evitado o aparecimento da DCV prematura, e no caso mais extremo a morte prematura como observado em algumas famílias. O diagnóstico e aconselhamento genético da FH é importante para a correcta percepção e prevenção do risco familiar de DCV. O estudo molecular fundamenta a instituição de terapêutica farmacológica adequada e a adopção de um estilo de vida saudável reduzindo substancialmente o risco cardiovascular. 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

Encapsulation and controlled release of bioactive compounds in lactoferrin-glycomacropeptide nanohydrogels : curcumin and caffeine as model compounds

Curcumin and caffeine (used as lipophilic and hydrophilic model compounds, respectively) were successfully encapsulated in lactoferrin-glycomacropeptide (Lf-GMP) nanohydrogels by thermal gelation showing high encapsulation efficiencies (>90 %). FTIR spectroscopy confirmed the encapsulation of bioactive compounds in Lf-GMP nanohydrogels and revealed that according to the encapsulated compound different interactions occur with the nanohydrogel matrix. The successful encapsulation of bioactive compounds in Lf-GMP nanohydrogels was also confirmed by fluorescence measurements and confocal laser scanning microscopy. TEM images showed that loaded nanohydrogels maintain their spherical shape with sizes of 112 and 126 nm for curcumin and caffeine encapsulated in Lf-GMP nanohydrogels, respectively; in both cases a polydispersity of 0.2 was obtained. The release mechanisms of bioactive compounds through Lf-GMP nanohydrogels were evaluated at pH 2 and pH 7, by fitting the Linear Superimposition Model to the experimental data. The bioactive compounds release was found to be pH-dependent: at pH 2, relaxation is the governing phenomenon for curcumin and caffeine compounds and at pH 7 Ficks diffusion is the main mechanism of caffeine release while curcumin was not released through Lf-GMP nanohydrogels

Portuguese Familial Hypercholesterolemia Study: presentation of the study and preliminary results

Familial hypercholesterolemia (FH) is an autosomal dominant genetic disorder caused, in the majority of cases, by a partial or total lack of functional low density lipoprotein receptors (LDLR). Mutations in the LDLR gene lead to increased plasma cholesterol levels, resulting in cholesterol deposition in the arteries, thereby increasing the risk of premature coronary heart disease. The homozygous form of FH is rare but heterozygous FH is common, although underdiagnosed in many populations, including the Portuguese. In 1999 the Portuguese Familial Hypercholesterolemia Study was begun at the National Institute of Health

Complex Phenotype of Hypercholesterolemia in a Family with Both ABCG8 and APOB Mutations

Familial hypercholesterolemia is a common genetic hypercholesterolemia caused by mutations in LDLR, APOB and PCSK9 that leads to premature atherosclerosis. Other rare disorders like sitosterolemia can present the same phenotype but have distinct therapeutic interventions. We present a case of severe hypercholesterolemia in a 5-year-old child found to have both familial hypercholesterolemia and sitosterolemia. The proband was diagnosed initially as familial hypercholesterolemia, but the lack of pathogenic variants with Sanger approach questioned this hypothesis. High levels of sitosterol established the diagnosis of sitosterolemia, genetically confirmed by an ABCG8 homozygous variant c.1974C>G/p. (Tyr658*). Next-generation sequencing re sequence for familial hypercholesterolemia genes revealed an APOB heterozygous functional variant (c.11477C>T/p. (Thr3826Met), in a region previously unstudied. The mother presented with the same genotype but a milder phenotype. Control of low-density lipoprotein cholesterol levels was only accomplished with dietary and therapeutic intervention for both sitosterolemia and familial hypercholesterolemia. The correct diagnosis of dyslipidemia is important to establish proper dietary and pharmacological intervention for atherosclerosis prevention.info:eu-repo/semantics/publishedVersio

Genetic diagnosis of familial hypercholesterolaemia: the importance of functional analysis of potential splice-site mutations

Familial hypercholesterolemia (FH) results from defective low-density lipoprotein receptor (LDLR) activity, mainly due to LDLR gene defects. Of the many different LDLR mutations found in patients with FH, about 6% of single base substitutions are located near or within introns, and are predicted to result in exon skipping, retention of an intron, or activation of cryptic sites during mRNA splicing. This paper reports on the Portuguese FH Study, which found 10 such mutations, 6 of them novel. For the mutations that have not been described before or those whose effect on function have not been analysed, their effect on splicing was investigated, using reverse transcriptase PCR analysis of LDLR mRNA from freshly isolated blood mononuclear cells. Two of these variants (c.313+6 T-->C, c.2389G-->T (p.V776L)) caused exon skipping, and one caused retention of an intron (c.1359-5C-->G), whereas two others (c.2140+5 G-->A and c.1061-8T-->C) had no apparent effect. Any effect of c.1185G-->C (p.V374V) on splicing could not be determined because it was on an allele with a promoter mutation (-42C-->G) that was probably not transcribed. Variants in four patients lost to follow-up could not be tested experimentally, but they almost certainly affect splicing because they disrupt the invariant AG or GT in acceptor (c.818-2A-->G) or donor (c.1060+1G-->A, c.1845+1delG and c.2547+1G-->A) spice sites. These findings emphasise that care must be taken before reporting the presence or absence of a splice-site mutation in the LDLR gene for diagnostic purposes. The study also shows that relatively simple, quick and inexpensive RNA assays can evaluate putative splicing mutations that are not always predictable by available software, thereby reducing genetic misdiagnosis of patients with FH