Isabel De Castro-Orós

Apolipoprotein E gene mutations in subjects with mixed hyperlipidemia and a clinical diagnosis of familial combined hyperlipidemia.

OBJECTIVEnRare mutations in the APOE gene, undetectable with the usual genotyping technique, are responsible for dominant familial dysbetalipoproteinemia (FD) and therefore could be easily misclassified as familial combined hyperlipidemia (FCHL). We aimed to identify APOE mutations associated with dominant combined hyperlipoproteinemia and to establish their frequency in subjects with a clinical diagnosis of FCHL.nnnMETHODS AND RESULTSnIn 279 unrelated subjects with FCHL in whom a functional LDLR mutation was excluded, sequencing of the entire APOE gene detected 9 carriers of a rare mutation: 5 subjects (1.8%) with the R136S mutation (arginine at residue 136 changed to serine) and 4 subjects (1.4%) with the p.Leu149del mutation, a 3-bp inframe deletion that results in the loss of leucine at position 149. Both genetic defects were detected with similar frequency (2.5% and 1.3%, respectively) in an independent group of 160 FCHL subjects from other locations in Spain. Family studies demonstrated cosegregation of these APOE mutations with hyperlipoproteinemia. R136S carriers showed dysbetalipoproteinemia, while the lipid phenotype of p.Leu149del carriers was IIa or IIb.nnnCONCLUSIONSnRare APOE mutations are responsible for approximately 3.5% of FCHL cases in our population. APOE R136S and p.Leu149del induce autosomal dominant FD and a phenotype indistinguishable from FCHL, respectively.

Promoter variant -204A > C of the cholesterol 7α-hydroxylase gene: association with response to plant sterols in humans and increased transcriptional activity in transfected HepG2 cells.

BACKGROUND & AIMSnThe bile acid pool influences intestinal cholesterol absorption because this process isxa0strictly dependent on micellar solubilization, which is disrupted by plant sterols (PS). Plasma lipid variation relates to promoter variant -204Axa0>xa0C (rs3808607) of the CYP7A1 gene encoding for 7α-hydroxylase, an enzyme for bile acid synthesis. We hypothesized that this polymorphism would be associated with variability in lipid responses to PS.nnnMETHODSnWe investigated 67 subjects (31 AA and 36 ACxa0+xa0CC) with lipid responses to PS documented in two studies. To assess the functionality of the -204Axa0>xa0C variant, electrophoretic mobility gel shift assays were performed and luciferase reporter plasmids containing the promoter were transfected into HepG2 cells.nnnRESULTSnCompared to AA-subjects, C-carriers showed significantly higher adjusted mean reductions in total cholesterol (0.14 versus 0.43xa0mmol/L, Pxa0=xa00.042) and increases in lathosterol-to-cholesterol ratios (0.10 versus 0.75, Pxa0=xa00.013). The C-construct caused a 78% promoter activity increase and gel-shift assays showed lower affinity for nuclear transcription factors, while in silico experiments predicted a binding site for inhibitory nuclear factors RXR-CAR.nnnCONCLUSIONSnResults suggest that promoter -204Axa0>xa0C variant is associated with enhanced CYP7A1 activity. Increased intestinal bile acids and ensuing more efficient cholesterol absorption might explain why C-allele carriers show enhanced cholesterol lowering and increased feedback cholesterol synthesis to PS intervention.

The genetic basis of familial hypercholesterolemia: inheritance, linkage, and mutations

Familial hypercholesterolemia (FH) is a genetic disorder of lipoprotein metabolism characterized by high plasma concentrations of low-density lipoprotein cholesterol (LDLc), tendon xanthomas, and increased risk of premature coronary heart disease. FH is one of the most common inherited disorders; there are 10,000,000 people with FH worldwide, mainly heterozygotes. The most common FH cause is mutations along the entire gene that encode for LDL receptor (LDLR) protein, but it has been also described that mutations in apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin type 9 genes produce this phenotype. About 17%–33% of patients with a clinical diagnosis of monogenic hypercholesterolemia do not harbor any genetic cause in the known loci. Because FH has been considered as a public health problem, it is very important for an early diagnosis and treatment. Recent studies have demonstrated the influence of the LDLR mutation type in the FH phenotype, associating a more severe clinical phenotype and worse advanced carotid artherosclerosis in patients with null than those with receptor-defective mutations. Since 2004, a molecular FH diagnosis based on a genetic diagnostic platform (Lipochip®; Progenika-Biopharma, Derio, Spain) has been developed. This analysis completes the adequate clinical diagnosis made by physicians. Our group has recently proposed new FH guidelines with the intention to facilitate the FH diagnosis. The treatment for this disease is based on the benefit of lowering LDLc and a healthy lifestyle. Actually, drug therapy is focused on using statins and combined therapy with ezetimibe and statins. This review highlights the recent progress made in genetics, diagnosis, and treatment for FH.

The p.Leu167del Mutation in APOE Gene Causes Autosomal Dominant Hypercholesterolemia by Down-regulation of LDL Receptor Expression in Hepatocytes

CONTEXTnThe p.Leu167del mutation in the APOE gene has been associated with hyperlipidemia.nnnOBJECTIVESnOur objective was to determine the frequency of p.Leu167del mutation in APOE gene in subjects with autosomal dominant hypercholesterolemia (ADH) in whom LDLR, APOB, and PCSK9 mutations had been excluded and to identify the mechanisms by which this mutant apo E causes hypercholesterolemia.nnnDESIGNnThe APOE gene was analyzed in a case-control study.nnnSETTINGnThe study was conducted at a University Hospital Lipid Clinic.nnnPATIENTS OR OTHER PARTICIPANTSnTwo groups (ADH, 288 patients; control, 220 normolipidemic subjects) were included.nnnINTERVENTIONnWe performed sequencing of APOE gene and proteomic and cellular experiments.nnnMAIN OUTCOME MEASUREnTo determine the frequency of the p.Leu167del mutation and the mechanism by which it causes hypercholesterolemia.nnnRESULTSnIn the ADH group, nine subjects (3.1%) were carriers of the APOE c.500_502delTCC, p.Leu167del mutation, cosegregating with hypercholesterolemia in studied families. Proteomic quantification of wild-type and mutant apo E in very low-density lipoprotein (VLDL) from carrier subjects revealed that apo E3 is almost a 5-fold increase compared to mutant apo E. Cultured cell studies revealed that VLDL from mutation carriers had a significantly higher uptake by HepG2 and THP-1 cells compared to VLDL from subjects with E3/E3 or E2/E2 genotypes. Transcriptional down-regulation of LDLR was also confirmed.nnnCONCLUSIONSnp.Leu167del mutation in APOE gene is the cause of hypercholesterolemia in the 3.1% of our ADH subjects without LDLR, APOB, and PCSK9 mutations. The mechanism by which this mutation is associated to ADH is that VLDL carrying the mutant apo E produces LDLR down-regulation, thereby raising plasma low-density lipoprotein cholesterol levels.

Common genetic variants contribute to primary hypertriglyceridemia without differences between familial combined hyperlipidemia and isolated hypertriglyceridemia.

Background—The majority of hypertriglyceridemias are diagnosed as familial combined hyperlipidemia (FCHL) and primary isolated hypertriglyceridemias. The contribution of common genetic variants in primary hypertriglyceridemias and the genetic difference between FCHL and isolated hypertriglyceridemias have not been thoroughly examined. Methods and Results—This study involved 580 patients with hypertriglyceridemias and 403 controls. Of the 37 single nucleotide polymorphisms examined, 12 located in 10 genes showed allelic and genotype frequency differences between hypertriglyceridemias and controls. The minor alleles of APOE, APOA5, GALNTN2, and GCKR variants were positively correlated with plasma triglycerides, whereas minor alleles of ADIPOR2, ANGPTL3, LPL, and TRIB1 polymorphisms were inversely associated. Body mass index, glucose, sex, rs328 and rs7007797 in LPL, rs662799 and rs3135506 in APOA5, and rs1260326 in GCKR explained 36% of the variability in plasma triglycerides, 7.3% of which was attributable to the genetic variables. LPL, GCKR, and APOA5 polymorphisms fit dominant, recessive, and additive inheritance models, respectively. Variants more frequently identified in isolated hypertriglyceridemias were rs7412 in APOE and rs1800795 in IL6; rs2808607 in CYP7A1 and rs3812316 and rs17145738 in MLXIPL were more frequent in FCHL. The other 32 single nucleotide polymorphisms presented similar frequencies between isolated hypertriglyceridemias and FCHL. Conclusions—Common genetic variants found in LPL, APOA5, and GCKR are associated with triglycerides levels in patients with primary hypertriglyceridemias. FCHL and isolated hypertriglyceridemias are probably trace to an accumulation of genetic variants predisposing to familial and sporadic hypertriglyceridemias or to hypertriglyceridemias and hypercholesterolemia in case of FCHL.

Functional analysis of LDLR promoter and 5' UTR mutations in subjects with clinical diagnosis of familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a dominant disorder due to mutations in the LDLR gene. Several mutations in the LDLR promoter are associated with FH. Screening of 3,705 Spanish FH patients identified 10 variants in the promoter and 5′ UTR. Here, we analyse the functionality of six newly identified LDLR variants. Mutations located in the LDLR promoter regulatory elements R2 and R3 (c.−155_‐150delACCCCinsTTCTGCAAACTCCTCCC, c.−136C>G, c.−140C>G, and c.−140C>T) resulted in 6 to 15% residual activity in reporter expression experiments and changes in nuclear protein binding affinity compared to wild type. No reduction was observed when cells were transfected with c.−208T, c.−88A, and c.−36G mutant fragments. Our results indicate that mutations localized in R2 and R3 are associated with hypercholesterolemia, whereas mutations outside the LDLR response elements are not a cause of FH. This data emphasizes the importance of functional analysis of variants in the LDLR promoter to determine their association with the FH phenotype.Hum Mutat 32:1–5, 2011.

APOA5 variants predispose hyperlipidemic patients to atherogenic dyslipidemia and subclinical atherosclerosis

BACKGROUNDnTriglycerides (TG) are the initiators of the metabolic changes leading to the atherogenic dyslipidemia, which is a major inducer of atherosclerosis as a result of quantitative and qualitative changes in lipoprotein subclass distributions. We hypothesized that variation at the of APOA5 gene locus, encoding apoAV, a key regulator of TG levels, significantly affect lipoprotein subclass distributions toward a more atherogenic pattern in both hyperTG patients and dyslipemic patients.nnnMETHODSnWe recruited four hundred and twenty-two subjects attending a Lipid Clinic, prior to lipid-lowering treatment. We genotyped two APOA5 variants, rs662799 (-1131T>C) and rs3135506 (S19W). Circulating lipoproteins were determined by nuclear magnetic resonance (NMR). Intima-media thickness (IMT) was evaluated using B-mode ultrasound.nnnRESULTSnCarriers of the rare alleles of rs662799 and rs3135506 compared to common allele homozygotes, had a significantly proatherogenic profile of the VLDL and LDL subclasses, resulting in increased concentrations of the proatherogenic subclasses, large VLDLs (+133%, p<0.001) and small LDLs (+34%, p=0.014). Significant changes in smaller HDL (+71%, p=0.032), as well as an 18% decrease in large HDL (p=0.046), were also been observed. This atherogenic NMR subclass distribution was significantly associated with increased carotid IMT. The observed effects were significantly stronger in patients with a BMI≥25 kg/m2 and in male and female patients with a waist circumference≥90 cm or ≥85 cm, respectively.nnnCONCLUSIONnIn a dyslipemic population, genetic variants of APOA5 modulate lipoprotein subclass distributions, inducing an atherogenic profile associated with IMT defined subclinical atherosclerosis.

Frequency of rare mutations and common genetic variations in severe hypertriglyceridemia in the general population of Spain

BackgroundHypertriglyceridemia (HTG) is a common complex metabolic trait that results of the accumulation of relatively common genetic variants in combination with other modifier genes and environmental factors resulting in increased plasma triglyceride (TG) levels. The majority of severe primary hypertriglyceridemias is diagnosed in adulthood and their molecular bases have not been fully defined yet. The prevalence of HTG is highly variable among populations, possibly caused by differences in environmental factors and genetic background. However, the prevalence of very high TG and the frequency of rare mutations causing HTG in a whole non-selected population have not been previously studied.MethodsThe total of 23,310 subjects over 18xa0years from a primary care-district in a middle-class area of Zaragoza (Spain) with TG >500xa0mg/dL were selected to establish HTG prevalence. Those affected of primary HTG were considered for further genetic analisys. The promoters, coding regions and exon-intron boundaries of LPL, LMF1, APOC2, APOA5, APOE and GPIHBP1 genes were sequenced. The frequency of rare variants identified was studied in 90 controls.ResultsOne hundred ninety-four subjects (1.04xa0%) had HTG and 90 subjects (46.4xa0%) met the inclusion criteria for primary HTG. In this subgroup, nine patients (12.3xa0%) were carriers of 7 rare variants in LPL, LMF1, APOA5, GPIHBP1 or APOE genes. Three of these mutations are described for the first time in this work. The presence of a rare pathogenic mutation did not confer a differential phenotype or a higher family history of HTG.ConclusionThe prevalence of rare mutations in candidate genes in subjects with primary HTG is low. The low frequency of rare mutations, the absence of a more severe phenotype or the dominant transmission of the HTG would not suggest the use of genetic analysis in the clinical practice in this population.

A genetic variant in the LDLR promoter is responsible for part of the LDL-cholesterol variability in primary hypercholesterolemia

BackgroundGWAS have consistently revealed that LDLR locus variability influences LDL-cholesterol in general population. Severe LDLR mutations are responsible for familial hypercholesterolemia (FH). However, most primary hypercholesterolemias are polygenic diseases. Although Cis-regulatory regions might be the cause of LDL-cholesterol variability; an extensive analysis of the LDLR distal promoter has not yet been performed. We hypothesized that genetic variants in this region are responsible for the LDLR association with LDL-cholesterol found in GWAS.MethodsFour-hundred seventy-seven unrelated subjects with polygenic hypercholesterolemia (PH) and without causative FH-mutations and 525 normolipemic subjects were selected. A 3103 pb from LDLR (-625 to +2468) was sequenced in 125 subjects with PH. All subjects were genotyped for 4 SNPs (rs17242346, rs17242739, rs17248720 and rs17249120) predicted to be potentially involved in transcription regulation by in silico analysis. EMSA and luciferase assays were carried out for the rs17248720 variant. Multivariable linear regression analysis using LDL-cholesterol levels as the dependent variable were done in order to find out the variables that were independently associated with LDL-cholesterol.ResultsThe sequencing of the 125 PH subjects did not show variants with minor allele frequencyu2009≥u200910%. The T-allele from g.3131Cu2009>u2009T (rs17248720) had frequencies of 9% (PH) and 16.4% (normolipemic), pu2009<u20090.00001. Studies of this variant with EMSA and luciferase assays showed a higher affinity for transcription factors and an increase of 2.5 times in LDLR transcriptional activity (T-allele vs C-allele). At multivariate analysis, this polymorphism with the lipoprotein(a) and age explainedu2009≈u200910% of LDL-cholesterol variability.ConclusionOur results suggest that the T-allele at the g.3131xa0Tu2009>u2009C SNP is associated with LDL-cholesterol levels, and explains part of the LDL-cholesterol variability. As a plausible cause, the T-allele produces an increase in LDLR transcriptional activity and lower LDL-cholesterol levels.

Energy-restricted, high-protein diets more effectively impact cardiometabolic profile in overweight and obese women than lower-protein diets

BACKGROUND & AIMSnHigh-protein energy-restricted diets have demonstrated efficacy in promoting weight loss in overweight and obesity. However, the protein percentage that achieves optimal efficacy and acceptability remains unknown. We sought to assess the effects of three energy-reduced diets with different percentages of calories from protein (20%, 27%, and 35%) on weight loss and lipids. Secondary outcomes included diet acceptability and compliance.nnnMETHODSnSix-month, randomized study included women aged 18-80 years with BMI of 27.5-45xa0kg/m2 and who were not taking lipid-lowering drugs. We randomly assigned 91 women to one of three calorie-reduced diets with: protein, 20%, 27%, or 35% (80% from animal protein); carbohydrates, 50%, 43%, or 35%; fat, 30%. Dietary intervention involved individual visits with a nutritionist every 2 weeks during the first 3 months. We performed a follow-up visit at 6 months.nnnRESULTSnEighty women aged 44.0xa0±xa09.08 years with BMI of 37.7xa0±xa03.39xa0kg/m2 completed the study. At 3 months, weight loss wasxa0-8.16xa0±xa04.18xa0kg,xa0-9.66xa0±xa05.28xa0kg, andxa0-10.7xa0±xa04.28xa0kg in the 20%, 27%, and 35%-protein groups, respectively (Pxa0=xa00.16). These figures slightly and homogeneously increased at 6 months. Around 65% of women following 35%-protein diet lost ≥10% of body weight vs. ∼33% in 20%-protein group (Pxa0=xa00.023). Significant decreases occurred in fat mass, lipids and insulin resistance, especially in the 35%-protein group (Pxa0<xa00.05 vs. 20% protein). This improvement was not fully explained by weight loss. Triglyceride change was negatively correlated with animal-protein intake. All groups provided similar responses to an acceptance, palatability, and satisfaction questionnaire.nnnCONCLUSIONSnAn energy-restricted diet with 35% protein, mostly of animal origin, more effectively impacts cardiometabolic profile than an energy-restricted diet with lower protein content although no clear benefit between diets in terms of overall weight loss was observed. The high-protein diet displayed an excellent safety profile and acceptability. This trial was registered in ClinicalTrials.gov as NCT02160496.nnnCLINICAL TRIAL REGISTRATIONnThe clinical trial has been registered in ClinicalTrials.gov (Identifier: NCT02160496).