LC-MS/MS analysis of plasma glucosylsphingosine as a biomarker for diagnosis and follow-up monitoring in Gaucher disease in the Spanish population.

Background Gaucher disease (GD), caused by a deficiency in acid ß-glucosidase, leads to the accumulation of glucosylsphingosine (GluSph), which has been used as a powerful biomarker for the diagnosis and follow-up of GD. Our aim was to perform the first retrospective study of GluSph in Spanish patients, analyzing its relationship with classical biomarkers and other parameters of disease and its utility regarding treatment monitoring. Methods Classical biomarkers were evaluated retrospectively by standard methods in a total of 145 subjects, including 47 GD patients, carriers, healthy controls and patients suffering from other lysosomal lipidoses. GluSph was also measured using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method developed as part of the present study. Results The optimized method presented intra- and inter-assay variations of 3.1 and 11.5%, respectively, overall recovery higher than 96% and linearity up to plasma concentrations of 1000 ng/mL with 100% specificity and sensitivity. Only GD patients displayed GluSph levels above 5.4 ng/mL at diagnosis and this was significantly correlated with the classical biomarkers chitotriosidase (r = 0.560) and the chemokine CCL18/PARC (CCL18/PARC) (ρ = 0.515), as well as with the Spanish magnetic resonance imaging index (S-MRI, r = 0.364), whereas chitotriosidase correlated with liver volume (r = 0.372) and CCL18/PARC increased in patients with bone manifestations (p = 0.005). GluSph levels decreased with treatment in naïve patients. Conclusions Plasma GluSph is the most disease-specific biomarker for GD with demonstrated diagnostic value and responsiveness to therapy. GluSph in the present series of patients failed to demonstrate better correlations with clinical characteristics at onset than classical biomarkers.

Single Nucleotide Variants Associated With Polygenic Hypercholesterolemia in Families Diagnosed Clinically With Familial Hypercholesterolemia.

INTRODUCTION AND OBJECTIVES: Approximately 20% to 40% of clinically defined familial hypercholesterolemia cases do not show a causative mutation in candidate genes, and some of them may have a polygenic origin. A cholesterol gene risk score for the diagnosis of polygenic hypercholesterolemia has been demonstrated to be valuable to differentiate polygenic and monogenic hypercholesterolemia. The aim of this study was to determine the contribution to low-density lipoprotein cholesterol (LDL-C) of the single nucleotide variants associated with polygenic hypercholesterolemia in probands with genetic hypercholesterolemia without mutations in candidate genes (nonfamilial hypercholesterolemia genetic hypercholesterolemia) and the genetic score in cascade screening in their family members. METHODS: We recruited 49 nonfamilial hypercholesterolemia genetic hypercholesterolemia families (294 participants) and calculated cholesterol gene scores, derived from single nucleotide variants in SORT1, APOB, ABCG8, APOE and LDLR and lipoprotein(a) plasma concentration. RESULTS: Risk alleles in SORT1, ABCG8, APOE, and LDLR showed a statistically significantly higher frequency in blood relatives than in the 1000 Genomes Project. However, there were no differences between affected and nonaffected members. The contribution of the cholesterol gene score to LDL-C was significantly higher in affected than in nonaffected participants (P = .048). The percentage of the LDL-C variation explained by the score was 3.1%, and this percentage increased to 6.9% in those families with the highest genetic score in the proband. CONCLUSIONS: Nonfamilial hypercholesterolemia genetic hypercholesterolemia families concentrate risk alleles for high LDL-C. Their contribution varies greatly among families, indicating the complexity and heterogeneity of these forms of hypercholesterolemias. The gene score explains a small percentage of LDL-C, which limits its use in diagnosis.

Functional analysis of new 3' untranslated regions genetic variants in genes associated with genetic hypercholesterolemias.

BACKGROUND: Familial hypercholesterolemia (FH) is the best-described autosomal dominant genetic hypercholesterolemia (GH). Mutations in candidate genes can explain a high proportion of FH cases, but for many, no causative mutations are detected (designed non-FG-GH), suggesting the existence of additional genetic variants associated with the disease. OBJECTIVE: We aimed to identify new single-nucleotide variants (SNVs) located at the 3' untranslated regions (3'UTRs) of the low-density lipoprotein receptor, low-density lipoprotein receptor-related protein-associated protein 1, ATP-binding cassette sub-family G member 5, and sterol regulatory element-binding protein 2 genes in non-FH-GH individuals and investigated whether the association of these SNVs with non-FH-GH could be explained by changes in the affinity of regulatory microRNAs (miRNA) targeting the sequences modified by the SNVs. METHODS: The study includes probands with non-FH-GH attending 2 lipid clinics in Spain. We performed functional analyses of selected variants using a luciferase reporter system. Through in silico target-prediction tools, we identified miRNAs, which binding to the 3'UTR could be affected by the presence of specific SNVs. We used analogs and inhibitors of these miRNAs to test this possibility. RESULTS: We identified 11 new SNVs showing significant association with non-FH-GH. We show that the presence of 4 of these SNVs leads to significant changes in the transcriptional levels of the reporter gene. Through mechanistic analysis, we identified 2 miRNAs (miR-27a and miR-133-3p) targeting the 3'UTR of sterol regulatory element-binding protein 2 and an additional miRNA (miR-92a) targeting the 3'UTR of low-density lipoprotein receptor-related protein-associated protein 1. CONCLUSION: Our findings reveal novel regulatory links between certain miRNAs and key genes regulating cholesterol homeostasis. They also highlight the potential of miRNAs as therapeutic targets for the treatment of FH.

Assessment of plasma chitotriosidase activity, CCL18/PARC concentration and NP-C suspicion index in the diagnosis of Niemann-Pick disease type C: a prospective observational study.

BACKGROUND: Niemann-Pick disease type C (NP-C) is a rare, autosomal recessive neurodegenerative disease caused by mutations in either the NPC1 or NPC2 genes. The diagnosis of NP-C remains challenging due to the non-specific, heterogeneous nature of signs/symptoms. This study assessed the utility of plasma chitotriosidase (ChT) and Chemokine (C-C motif) ligand 18 (CCL18)/pulmonary and activation-regulated chemokine (PARC) in conjunction with the NP-C suspicion index (NP-C SI) for guiding confirmatory laboratory testing in patients with suspected NP-C. METHODS: In a prospective observational cohort study, incorporating a retrospective determination of NP-C SI scores, two different diagnostic approaches were applied in two separate groups of unrelated patients from 51 Spanish medical centers (n = 118 in both groups). From Jan 2010 to Apr 2012 (Period 1), patients with ≥2 clinical signs/symptoms of NP-C were considered 'suspected NP-C' cases, and NPC1/NPC2 sequencing, plasma chitotriosidase (ChT), CCL18/PARC and sphingomyelinase levels were assessed. Based on findings in Period 1, plasma ChT and CCL18/PARC, and NP-C SI prediction scores were determined in a second group of patients between May 2012 and Apr 2014 (Period 2), and NPC1 and NPC2 were sequenced only in those with elevated ChT and/or elevated CCL18/PARC and/or NP-C SI ≥70. Filipin staining and 7-ketocholesterol (7-KC) measurements were performed in all patients with NP-C gene mutations, where possible. RESULTS: In total across Periods 1 and 2, 10/236 (4%) patients had a confirmed diagnosis o NP-C based on gene sequencing (5/118 [4.2%] in each Period): all of these patients had two causal NPC1 mutations. Single mutant NPC1 alleles were detected in 8/236 (3%) patients, overall. Positive filipin staining results comprised three classical and five variant biochemical phenotypes. No NPC2 mutations were detected. All patients with NPC1 mutations had high ChT activity, high CCL18/PARC concentrations and/or NP-C SI scores ≥70. Plasma 7-KC was higher than control cut-off values in all patients with two NPC1 mutations, and in the majority of patients with single mutations. Family studies identified three further NP-C patients. CONCLUSION: This approach may be very useful for laboratories that do not have mass spectrometry facilities and therefore, they cannot use other NP-C biomarkers for diagnosis.

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

BACKGROUND & AIMS: High-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. METHODS: Six-month, randomized study included women aged 18-80 years with BMI of 27.5-45 kg/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. RESULTS: Eighty women aged 44.0 ± 9.08 years with BMI of 37.7 ± 3.39 kg/m2 completed the study. At 3 months, weight loss was -8.16 ± 4.18 kg, -9.66 ± 5.28 kg, and -10.7 ± 4.28 kg in the 20%, 27%, and 35%-protein groups, respectively (P = 0.16). 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 (P = 0.023). Significant decreases occurred in fat mass, lipids and insulin resistance, especially in the 35%-protein group (P < 0.05 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. CONCLUSIONS: An 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. CLINICAL TRIAL REGISTRATION: The clinical trial has been registered in ClinicalTrials.gov (Identifier: NCT02160496).

Rare genetic variants with large effect on triglycerides in subjects with a clinical diagnosis of familial vs nonfamilial hypertriglyceridemia.

BACKGROUND: Most primary severe hypertriglyceridemias (HTGs) are diagnosed in adults, but their molecular foundations have not been completely elucidated. OBJECTIVE: We aimed to identify rare dysfunctional mutations in genes encoding regulators of lipoprotein lipase (LPL) function in patients with familial and non-familial primary HTG. METHODS: We sequenced promoters, exons, and exon-intron boundaries of LPL, APOA5, LMF1, and GPIHBP1 in 118 patients with severe primary HTG (triglycerides >500 mg/dL) and 53 normolipidemic controls. Variant functionality was analyzed using predictive software and functional assays for mutations in regulatory regions. RESULTS: We identified 29 rare variants, 10 of which had not been previously described: c.(-16A>G), c.(1018+2G>A), and p.(His80Arg) in LPL; p.(Arg143Alafs*57) in APOA5; p.(Val140Ile), p.(Leu235Ile), p.(Lys520*), and p.(Leu552Arg) in LMF1; and c.(-83G>A) and c.(-192A>G) in GPIHBP1. The c.(1018+2G>A) variant led to deletion of exon 6 in LPL cDNA, whereas the c.(-16A>G) analysis showed differences in the affinity for nuclear proteins. Overall, 20 (17.0%) of the patients carried at least one allele with a rare pathogenic variant in LPL, APOA5, LMF1, or GPIHBP1. The presence of a rare pathogenic variant was not associated with lipid values, family history of HTG, clinical diagnosis, or previous pancreatitis. CONCLUSIONS: Less than one in five subjects with triglycerides >500 mg/dL and no major secondary cause for HTG may carry a rare pathogenic mutation in LPL, APOA5, LMF1, or GPIHBP1. The presence of a rare pathogenic variant is not associated with a differential phenotype.

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

BACKGROUND: Hypertriglyceridemia (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. METHODS: The total of 23,310 subjects over 18 years from a primary care-district in a middle-class area of Zaragoza (Spain) with TG >500 mg/dL were selected to establish HTG prevalence. Those affected of primary HTG were considered for further genetic analysis. 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. RESULTS: One hundred ninety-four subjects (1.04%) had HTG and 90 subjects (46.4%) met the inclusion criteria for primary HTG. In this subgroup, nine patients (12.3%) 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. CONCLUSION: The 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.

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

CONTEXT: The p.Leu167del mutation in the APOE gene has been associated with hyperlipidemia. OBJECTIVES: Our 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. DESIGN: The APOE gene was analyzed in a case-control study. SETTING: The study was conducted at a University Hospital Lipid Clinic. PATIENTS OR OTHER PARTICIPANTS: Two groups (ADH, 288 patients; control, 220 normolipidemic subjects) were included. INTERVENTION: We performed sequencing of APOE gene and proteomic and cellular experiments. MAIN OUTCOME MEASURE: To determine the frequency of the p.Leu167del mutation and the mechanism by which it causes hypercholesterolemia. RESULTS: In 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. CONCLUSIONS: p.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.

Genetic Variants of LDLR and PCSK9 Associated with Variations in Response to Antihypercholesterolemic Effects of Armolipid Plus with Berberine.

BACKGROUND: Armolipid Plus (AP) is a nutraceutical that contains policosanol, fermented rice with red yeast, berberine, coenzyme Q10, folic acid, and astaxanthin. It has been shown to be effective in reducing plasma LDL cholesterol (LDLc) levels. In the multicenter randomized trial NCT01562080, there was large interindividual variability in the plasma LDLc response to AP supplementation. We hypothesized that the variability in LDLc response to AP supplementation may be linked to LDLR and PCSK9 polymorphisms. MATERIAL AND METHODS: We sequenced the LDLR 3' and 5' untranslated regions (UTR) and the PCSK9 5' UTR of 102 participants with moderate hypercholesterolemia in trial NCT01562080. In this trial, 50 individuals were treated with AP supplementation and the rest with placebo. RESULTS: Multiple linear regression analysis, using the response of LDLc levels to AP as the dependent variable, revealed that polymorphisms rs2149041 (c.-3383C>G) in the PCSK9 5' UTR and rs14158 (c.*52G>A) in the LDLR 3' UTR explained 14.1% and 6.4%, respectively, of the variability after adjusting for gender, age, and BMI of individuals. Combining polymorphisms rs2149041 and rs14158 explained 20.5% of this variability (p < 0.004). CONCLUSIONS: Three polymorphisms in the 3' UTR region of LDLR, c.*52G>A, c.*504G>A, and c.*773A>G, and two at the 5' UTR region of PCSK9, c.-3383C>G and c.-2063A>G, were associated with response to AP. These results could explain the variability observed in the response to berberine among people with moderate hypercholesterolemia, and they may be useful in identifying patients who could potentially benefit from supplementation with AP.

Cosegregation of serum cholesterol with cholesterol intestinal absorption markers in families with primary hypercholesterolemia without mutations in LDLR, APOB, PCSK9 and APOE genes.

BACKGROUND AND AIM: The genetic cause and pathogenic mechanism of approximately 20-40% of autosomal dominant hypercholesterolemias (ADH) are unknown. Increased cholesterol intestinal absorption has been associated to ADH. If this variation contributes to their pathogenesis is unknown. METHODS AND RESULTS: We studied cholesterol absorption (phytosterols and cholestanol serum concentrations) and cholesterol synthesis (desmosterol serum concentration) in 20 families with ADH without causal mutations in LDLR, APOB, PCSK9 or APOE genes (non-FH ADH) selected from 54 non-FH ADH probands with (non-cholesterol sterol concentrations above 75th percentile) and without (under 75th percentile) hyperabsorption. The concentrations of cholestanol, sitosterol, campesterol and stigmasterol were higher in affected than in non-affected subjects (p = 0.003, <0.001.<0.001, 0.002, respectively). There was a strong cosegregation of hyperabsorption with high LDL cholesterol within hyperabsorber families with odds ratio 6.80 (confidence interval 1.656-27.9), p = 0.008. In hyperabsorber families, 60.5% of subjects were hyperabsorbers and 76% of them had high LDL cholesterol versus 38.3% and 63% in non-hyperabsorber families, respectively. CONCLUSION: Most hypercholesterolemic family members with a hyperabsorber proband are hyperabsorbers. These absorption markers are significantly and positively associated with LDL cholesterol, and predispose to high LDL cholesterol in family members. Our data suggest that complex interindividual variation in cholesterol absorption is involved in many non-FH ADH.

APOA5 variants predispose hyperlipidemic patients to atherogenic dyslipidemia and subclinical atherosclerosis.

BACKGROUND: Triglycerides (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. METHODS: We 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. RESULTS: Carriers 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. CONCLUSION: In a dyslipemic population, genetic variants of APOA5 modulate lipoprotein subclass distributions, inducing an atherogenic profile associated with IMT defined subclinical atherosclerosis.

[Identification of variants in LMF1 gene associated with primary hypertriglyceridemia]. / Identificación de variantes en el gen LMF1 asociadas con hipertrigliceridemia primaria.

The majority of severe primary hypertriglyceridemia (HTG) are diagnosed in adults, and their molecular bases have not yet been fully defined. The promoter, coding regions and intron-exon boundaries of LMF1 were sequenced in 112 patients with severe primary hipertrigliceridemia (defined as TG above 500mg/dl). Five patients (4.46%) were carriers of four rare variants in the LMF1 gene associated with HTG, which participate in lipoprotein lipase (LpL) function. Also, we have identified two common variants, c.194-28 T>G and c.729+18C>G that were associated with HTG, with a different allelic frequency to that observed in the general population. A bioinformatic analysis of all found variants was conducted, defining the following as potentially harmful: p.Arg364Gln, p.Arg451Trp, p.Pro562Arg and p.Leu85Leu. Our results suggest that LMF1 mutations are involved in a substantial proportion of cases with severe HTG, putting together the moderate-aggressive effect of rare mutations with polymorphisms classically associated with this disease.

Serum plant sterols as surrogate markers of dietary compliance in familial dyslipidemias.

BACKGROUND & AIMS: A well-balanced diet is the first-line treatment in hyperlipidemia. The objective was to study the association between serum phytosterols and dietary patterns to use them as surrogate markers of dietary compliance in primary dyslipidemias. METHODS: 288 patients with primary hyperlipidemias (192 autosomal dominant hypercholesterolemia (ADH) and 96 familial combined hyperlipidemia (FCHL)) were included. Principal factor analysis identified 2 major dietary patterns using a 137-item food frequency questionnaire. "Vegetable & Fruits pattern" was characterized by higher intake of fruits, green beans, nuts, tomatoes, roasted or boiled potatoes, lettuce and chard and lower of processed baked goods, pizza and beer. "Western pattern" was positively characterized by hamburgers, pasta, sunflower oil, rice, chickpeas, whole milk, veal, red beans and negatively with white fish. Serum non-cholesterol sterols were determined by HPLC-MS/MS. RESULTS: Plant sterols to-total cholesterol (TC) levels were lower with a higher adherence to a "Vegetable & Fruits pattern" (P = 0.009), mainly in ADH subjects (R(2) = 0.019). Their concentration was greater with higher compliance to "Western pattern" especially in FCHL (P = 0.014). Higher levels of synthesis markers-to-TC with a greater adherence to "Vegetable & Fruits pattern" were found (P = 0.001) (R(2) = 0.033 and R(2) = 0.109 in ADH and FCHL respectively). CONCLUSION: In subjects with primary dislipidemia, dietary patterns associate with serum absorption and synthesis markers, but no with lipid concentrations. The influence of diet on non-cholesterol sterols levels is not powerful enough to use them as subrogate markers.

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.

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

BACKGROUND: GWAS 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. METHODS: Four-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. RESULTS: The sequencing of the 125 PH subjects did not show variants with minor allele frequency ≥ 10%. The T-allele from g.3131C > T (rs17248720) had frequencies of 9% (PH) and 16.4% (normolipemic), p < 0.00001. 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 explained ≈ 10% of LDL-cholesterol variability. CONCLUSION: Our results suggest that the T-allele at the g.3131 T > C 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.

Bile acid synthesis precursors in familial combined hyperlipidemia: the oxysterols 24S-hydroxycholesterol and 27-hydroxycholesterol.

Familial combined hyperlipidemia (FCHL), the most common inherited disorder of lipid metabolism is characterized by increasing cholesterol synthesis precursors due to hepatic overproduction of cholesterol. The bile acids synthesis pathway has not been previously studied in FCHL. The aim of this work was to study the oxysterol levels which are involved in the bile acids synthesis from cholesterol in FCHL. Clinical parameters and subclinical atherosclerosis were studied in a total of 107 FCHL patients and 126 normolipidemic controls. Non cholesterol sterols (desmosterol and lanosterol) and oxysterols (27-hydroxycholesterol and 24S-hydroxycholesterol) were measured by high performance liquid chromatography tandem mass spectrometry. Desmosterol and lanosterol, markers of cholesterol synthesis, had a positive correlation with BMI and apo B. However, no correlation was found for 24S-hydroxycholesterol and 27-hydroxycholesterol, precursors of bile acids, with these clinical parameters. Only 27-hydroxycholesterol had a positive correlation with apo B, ρ=0.204 (P=0.037). All oxysterol levels were higher in FHCL as compared to normal controls. A total of 59 FCHL subjects (59%) presented values of 24S-hydroxycholesterol above the 95th percentile of this oxysterol in the control population. All oxysterols showed no association with fat mass in contrast with non-cholesterol sterols. FCHL subjects with oxysterol overproduction had less carotid intima media thickness (cIMT), which suggests less atherosclerosis in these subjects. In summary, our data indicate that high oxysterol levels might be good markers of FCHL, unrelated to fat mass, and may exert a protective mechanism for cholesterol accumulation.

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

OBJECTIVE: Rare 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. METHODS AND RESULTS: In 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. CONCLUSIONS: Rare 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.

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.

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 & AIMS: The bile acid pool influences intestinal cholesterol absorption because this process is strictly dependent on micellar solubilization, which is disrupted by plant sterols (PS). Plasma lipid variation relates to promoter variant -204A > C (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. METHODS: We investigated 67 subjects (31 AA and 36 AC + CC) with lipid responses to PS documented in two studies. To assess the functionality of the -204A > C variant, electrophoretic mobility gel shift assays were performed and luciferase reporter plasmids containing the promoter were transfected into HepG2 cells. RESULTS: Compared to AA-subjects, C-carriers showed significantly higher adjusted mean reductions in total cholesterol (0.14 versus 0.43 mmol/L, P = 0.042) and increases in lathosterol-to-cholesterol ratios (0.10 versus 0.75, P = 0.013). 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. CONCLUSIONS: Results suggest that promoter -204A > C 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.