Identification and validation of seven new loci showing differential DNA methylation related to serum lipid profile: an epigenome-wide approach. The REGICOR study.

Lipid traits (total, low-density and high-density lipoprotein cholesterol, and triglycerides) are risk factors for cardiovascular disease. DNA methylation is not only an inherited but also modifiable epigenetic mark that has been related to cardiovascular risk factors. Our aim was to identify loci showing differential DNA methylation related to serum lipid levels. Blood DNA methylation was assessed using the Illumina Human Methylation 450 BeadChip. A two-stage epigenome-wide association study was performed, with a discovery sample in the REGICOR study (n = 645) and validation in the Framingham Offspring Study (n = 2,542). Fourteen CpG sites located in nine genes (SREBF1, SREBF2, PHOSPHO1, SYNGAP1, ABCG1, CPT1A, MYLIP, TXNIP and SLC7A11) and 2 intergenic regions showed differential methylation in association with lipid traits. Six of these genes and 1 intergenic region were new discoveries showing differential methylation related to total cholesterol (SREBF2), HDL-cholesterol (PHOSPHO1, SYNGAP1 and an intergenic region in chromosome 2) and triglycerides (MYLIP, TXNIP and SLC7A11). These CpGs explained 0.7%, 9.5% and 18.9% of the variability of total cholesterol, HDL cholesterol and triglycerides in the Framingham Offspring Study, respectively. The expression of the genes SREBF2 and SREBF1 was inversely associated with methylation of their corresponding CpGs (P-value = 0.0042 and 0.0045, respectively) in participants of the GOLDN study (n = 98). In turn, SREBF1 expression was directly associated with HDL cholesterol (P-value = 0.0429). Genetic variants in SREBF1, PHOSPHO1, ABCG1 and CPT1A were also associated with lipid profile. Further research is warranted to functionally validate these new loci and assess the causality of new and established associations between these differentially methylated loci and lipid metabolism.

Different protein composition of low-calorie diet differently impacts adipokine profile irrespective of weight loss in overweight and obese women.

BACKGROUND AND AIMS: High-protein (HP) diets have shown benefits in cardiometabolic markers such as insulin or triglycerides but the responsible mechanisms are not known. We aimed to assess the effect of three energy-restricted diets with different protein contents (20%, 27%, and 35%; ∼80% coming from animal source) on plasma adipokine concentration and its association with changes in cardiometabolic markers. METHODS: Seventy-six women (BMI 32.8 ± 2.93) were randomized to one of three calorie-reduced diets, with protein, 20%, 27%, or 35%; carbohydrates, 50%, 43%, or 35%; and fat, 30%, for 3 months. Plasma adipokine (leptin, resistin, adiponectin, and retinol-binding protein 4; RBP4) levels were assessed. RESULTS: After 3 months, leptin concentration decreased in all groups without differences among them, while resistin levels remained unchanged. Adiponectin concentration heterogeneously changed in all groups (P for trend = 0.165) and resistin concentration did not significantly change. RPB4 significantly decreased by -17.5% (-31.7, -3.22) in 35%-protein diet (P for trend = 0.024 among diets). Triglycerides improved in women following the 35%-protein diet regardless of weight loss; RBP4 variation significantly influenced triglyceride concentration change by 24.9% and 25.9% when comparing 27%- and 35%- with 20%-protein diet, respectively. CONCLUSIONS: A 35%-protein diet induced a decrease in RBP4 regardless of weight loss, which was directly associated with triglyceride concentration improvement. These findings suggest that HP diets improve the cardiometabolic profile, at least in part, through changes in adipokine secretion. Whether this beneficial effect of HP diet is due to improvements in hepatic or adipose tissue functionality should be elucidated. CLINICAL TRIAL REGISTRATION: The clinical trial has been registered in ClinicalTrials.gov (Identifier: NCT02160496).

Effect of different fat-enriched meats on non-cholesterol sterols and oxysterols as markers of cholesterol metabolism: Results of a randomized and cross-over clinical trial.

BACKGROUND AND AIM: Different kinds of fatty acids can affect the synthesis, absorption, and elimination of cholesterol. This study was carried out to assess the associations of cholesterol metabolism with the intake of two meats with different fatty acid composition in healthy volunteers. METHODS AND RESULTS: The study group was composed of 20 subjects (12 males and eight females; age, 34.4 ± 11.6 years; body mass index (BMI), 23.5 ± 2.3 kg/m(2); low-density lipoprotein (LDL) cholesterol, 2.97 ± 0.55 mmol/l; high-density lipoprotein (HDL) cholesterol, 1.61 ± 0.31 mmol/l; triglycerides (TG), 1.06 ± 0.41 mmol/l) who completed a 30-day randomized and cross-over study to compare the cholesterol metabolism effect of 250 g of low-fat lamb versus 250 g of high-fat lamb per day in their usual diet. Cholesterol absorption, synthesis, and elimination were estimated from the serum non-cholesterol sterol and oxysterol concentrations analyzed by a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). No changes in weight, plasma lipids, or physical activity were observed across the study. Cholesterol intestinal absorption was decreased with both diets. Cholesterol synthesis and elimination decreased during the low-fat lamb dietary intervention (ρ = 0.048 and ρ = 0.005, respectively). CONCLUSION: Acute changes in the diet fat content modify the synthesis, absorption, and biliary elimination of cholesterol. These changes were observed even in the absence of total and LDL cholesterol changes in plasma. REGISTRATION NUMBER FOR CLINICAL TRIALS: ClinicalTrials.gov PRS, NCT02259153.

Carotid atherosclerosis and lipoprotein particle subclasses in familial hypercholesterolaemia and familial combined hyperlipidaemia.

BACKGROUND AND AIMS: Familial hypercholesterolaemia (FH) and familial combined hyperlipidaemia (FCH) are common atherogenic disorders with great variability in cardiovascular disease (CVD). No direct atherosclerosis burden comparisons have been performed between FH and FCH in relation to lipoprotein particle distribution. METHODS AND RESULTS: Risk factors and three measures of carotid intima-media thickness (IMT) in both sides were determined in 572 FH, 250 FCH and 200 controls. Lipoproteins were assessed by nuclear magnetic resonance (NMR) spectroscopy. Compared with controls, IMT measures were increased in FH and FCH. FCH had the highest adjusted mean-maximum IMT. FH had twice low-density lipoprotein (LDL) particles than controls, but similar LDL subclass size and distribution. FCH subjects also had increased LDL particles and the highest number of small LDL (1519 ± 731 nmol l(-1) vs. 887 ± 784 nmol l(-1) in FH and 545 ± 409 nmol l(-1) in controls). Age, gender, cholesterol/high-density lipoprotein (HDL) ratio, smoking and systolic blood pressure were independently associated with IMT in FH (r(2) = 0.38). The same variables, except cholesterol/HDL ratio, were associated with IMT in FCH (r(2) = 0.40). Among NMR lipoproteins, only VLDL and chylomicrons increased IMT prediction in FCH by 0.8%. CONCLUSION: FH and FCH subjects show increased carotid atherosclerosis in relation to classical risk factors. Lipoprotein subclasses do not substantially contribute to IMT variability.

Metabolic syndrome and coronary heart disease among Spanish male workers: a case-control study of MESYAS.

BACKGROUND AND AIMS: In Spain, the incidence of coronary heart disease is below that expected based on the burden of classic cardiovascular risk factors present in the population. Whether the risk associated with metabolic syndrome is lower in Spain deserves to be investigated. This study evaluates the association of incident clinical coronary heart disease with metabolic syndrome and each of its individual defining components in a sample of Spanish working males. METHODS AND RESULTS: Among the workers of a factory (MESYAS registry), 208 incident cases of coronary heart disease (between 1981 and 2005) were age-matched with 2080 healthy workers visited in 2004-2005. Metabolic syndrome was characterized using modified criteria of the joint consensus definition (2009). Metabolic syndrome was strongly associated with coronary heart disease (OR = 4.03; 95% CI: 2.98, 5.45) and the risk seemed to be fully explained by metabolic syndrome components (OR = 0.84, p = 0.54 after adjustment). Odds ratios for the independent effects of the diagnostic criteria were: hypertriglyceridemia (OR = 3.39, p < 0.001), hyperglycemia (OR = 2.70, p < 0.001), low HDL cholesterol (OR = 2.35, p < 0.001), hypertension (OR = 1.49, p = 0.016) and overweight (OR = 1.07, p = 0.678). Young workers showed a higher risk associated with metabolic syndrome. CONCLUSION: The risk associated with metabolic syndrome is fully explained by its components considered independently. The risk of coronary heart disease in a Spanish male working population is considerably increased among those with metabolic syndrome, by a factor similar to that described for other countries. Public health measures to prevent a rise in the prevalence of metabolic syndrome are advisable to minimize cardiovascular disease rate in Spain.

A presumptive new locus for autosomal dominant hypercholesterolemia mapping to 8q24.22.

Molecular testing of patients with autosomal dominant hypercholesterolemia (ADH) fails to detect a causal functional mutation in 15.25% of subjects. We studied an ADH pedigree in which known ADH-causing genes (LDLR, APOB and PCSK9) were excluded. Genome-wide analysis on 15 family members detected significant association for ADH and dbSNP RS ID rs965814 (G/A), located in 8q24.22 cytoband. ADH was significantly associated to rs965814 G allele (p < 0.05) in a case-control study based on 200 unrelated ADH subjects without LDLR or APOB gene defects and 198 normolipidemic controls. We chose 24 markers for a detailed analysis of 8q24.22 cytoband, now based on an extended set of family members (21 individuals). One particular 24 marker haplotype was significantly associated to both higher total and low-density lipoprotein-cholesterol concentrations. Similar results were found for a shorter haplotype, composed of the distal six markers from the complete haplotype. Therefore, a presumptive new locus for ADH could be located in 8q24.22 cytoband, a region not previously linked or associated to ADH.

Association of plasma markers of cholesterol homeostasis with metabolic syndrome components. A cross-sectional study.

BACKGROUND AND AIMS: Increased plasma phytosterols, which reflect enhanced cholesterol absorption, have been related to an increased risk of cardiovascular disease (CVD). However, high CVD risk conditions, such as obesity, diabetes and the metabolic syndrome (MetS) have been associated with reduced cholesterol absorption. We investigated associations between plasma noncholesterol sterols and MetS components. METHODS AND RESULTS: With a cross-sectional design, we related MetS components to plasma noncholesterol sterol-to-cholesterol ratios measured by gas chromatography in 674 dyslipidemic patients and 361 healthy subjects participating in a prospective cohort study. Plasma phytosterol-to-cholesterol ratios were inversely associated with all components of the MetS. In the dyslipidemic group, multivariable analyses showed that a 1-SD increase in sitosterol-to-cholesterol ratio was associated with a reduced risk for any MetS feature, ranging from 0.57 (95% CI, 0.45 to 0.71) for visceral adiposity to 0.82 (95% CI, 0.69 to 0.98) for high blood pressure. The risk of having MetS was nearly halved, with ORs of 0.49 (95% CI, 0.38 to 0.64) or 0.56 (95% CI, 0.44-0.70), depending on the definition. Results were opposed for plasma lathosterol, a marker of cholesterol synthesis. Most findings were reproduced in the healthy cohort. ApoE genotype was unrelated to plasma noncholesterol sterols. CONCLUSION: In both dyslipidemic and healthy populations, MetS is associated with increased plasma lathosterol, a cholesterol synthesis marker, and decreased plasma sitosterol, a marker of cholesterol absorption. Elevated plasma phytosterols related to a lower frequency of cardiometabolic risk factors, suggesting that they are associated with a reduced CVD risk.

ANGPTL3 gene variants in subjects with familial combined hyperlipidemia.

Angiopoietin-like 3 (ANGPTL3) plays an important role in lipid metabolism in humans. Loss-of-function variants in ANGPTL3 cause a monogenic disease named familial combined hypolipidemia. However, the potential contribution of ANGPTL3 gene in subjects with familial combined hyperlipidemia (FCHL) has not been studied. For that reason, the aim of this work was to investigate the potential contribution of ANGPTL3 in the aetiology of FCHL by identifying gain-of-function (GOF) genetic variants in the ANGPTL3 gene in FCHL subjects. ANGPTL3 gene was sequenced in 162 unrelated subjects with severe FCHL and 165 normolipemic controls. Pathogenicity of genetic variants was predicted with PredictSNP2 and FruitFly. Frequency of identified variants in FCHL was compared with that of normolipemic controls and that described in the 1000 Genomes Project. No GOF mutations in ANGPTL3 were present in subjects with FCHL. Four variants were identified in FCHL subjects, showing a different frequency from that observed in normolipemic controls: c.607-109T>C, c.607-47_607-46delGT, c.835+41C>A and c.*52_*60del. This last variant, c.*52_*60del, is a microRNA associated sequence in the 3'UTR of ANGPTL3, and it was present 2.7 times more frequently in normolipemic controls than in FCHL subjects. Our research shows that no GOF mutations in ANGPTL3 were found in a large group of unrelated subjects with FCHL.

Haplotype analyses, mechanism and evolution of common double mutants in the human LDL receptor gene.

Familial hypercholesterolemia (FH), an autosomal dominant inherited disorder resulting in increased levels of circulating plasma low-density lipoprotein (LDL), tendon xanthomas and premature coronary artery disease (CAD), is caused by defects in the LDL receptor gene (LDLR). Three widespread LDLR alterations not causing FH (c.1061-8T>C, c.2177C>T and c.829G>A) and one mutation (c.12G>A) with narrow geographical distribution and thought to cause disease were investigated. In an attempt to improve knowledge on their origin, spread and possible selective effects, estimations of the ages of these variants (t generations) and haplotype analysis were performed by genotyping 86 healthy individuals and 98 FH patients in Spain for five LDLR SNPs: c.81T>C, c.1413G>A, c.1725C>T, c.1959T>C, and c.2232G>A; most patients carried two of these LDLR variants simultaneously. It was found that both the c.1061-8T>C (t = 54) and c.2177C>T alterations (t = 62) arose at about the same time (54 and 62 generations ago, respectively) in the CGCTG haplotype, while the c.12G>A mutation (t = 70) appeared in a CGCCG haplotype carrying an earlier c.829G>A alteration (t = 83). The estimated ages of selectively neutral alterations could explain their distribution by migrations. The origin of the c.12G>A mutation could be in the Iberian Peninsula; despite its estimated age, a low selective pressure could explain its conservation in Spain from where it could have spread to China and Mexico, since the sixteenth century through the Spanish/Portuguese colonial expeditions.

An NPC1L1 gene promoter variant is associated with autosomal dominant hypercholesterolemia.

BACKGROUND AND AIMS: A substantial number of subjects with autosomal dominant hypercholesterolemia (ADH) do not have LDL receptor (LDLR) or apolipoprotein B (APOB) mutations. Some ADH subjects appear to hyperabsorb sterols from the intestine, thus we hypothesized that they could have variants of the Niemann-Pick C1-Like 1 gene (NPC1L1). NPC1L1 encodes a crucial protein involved in intestinal sterol absorption. METHODS AND RESULTS: Four NPC1L1 variants (-133A>G, -18C>A, 1679C>G, 28650A>G) were analyzed in 271 (155 women and 116 men) ADH bearers without mutations in LDLR or APOB aged 30-70years and 274 (180 women and 94 men) control subjects aged 25-65years. The AC haplotype determined by the -133A>G and -18C>A variants was underrepresented in ADH subjects compared to controls (p=0.01). In the ADH group, cholesterol absorption/synthesis markers were significantly lower in AC homozygotes that in all others haplotypes. Electrophoretic mobility shift assay (EMSA) results revealed that the -133A-specific oligonucleotide produced a retarded band stronger than the -133G allele. Luciferase activity with NPC1L1 -133G variant was 2.5-fold higher than with the -133A variant. CONCLUSION: The -133A>G polymorphism exerts a significant effect on NPC1L1 promoter activity. NPC1L1 promoter variants might explain in part the hypercholesterolemic phenotype of some subjects with nonLDLR/nonAPOB ADH.

Replacement of cysteine at position 46 in the first cysteine-rich repeat of the LDL receptor impairs apolipoprotein recognition.

BACKGROUND AND AIMS: Pathogenic mutations in the Low Density Lipoprotein Receptor gene (LDLR) cause Familial Hypercholesterolemia (FH), one of the most common genetic disorders with a prevalence as high as 1 in 200 in some populations. FH is an autosomal dominant disorder of lipoprotein metabolism characterized by high blood cholesterol levels, deposits of cholesterol in peripheral tissues such as tendon xanthomas and accelerated atherosclerosis. To date, 2500 LDLR variants have been identified in the LDLR gene; however, only a minority of them have been experimentally characterized and proven to be pathogenic. Here we investigated the role of Cys46 located in the first repeat of the LDL receptor binding domain in recognition of apolipoproteins. METHODS: Activity of the p.(Cys46Gly) LDLR variant was assessed by immunoblotting and flow cytometry in CHO-ldlA7 expressing the receptor variant. Affinity of p.(Cys46Gly) for LDL and VLDL was determined by solid-phase immunoassays and in silico analysis was used to predict mutation effects. RESULTS AND CONCLUSION: Functional characterization of p.(Cys46Gly) LDLR variant showed impaired LDL and VLDL binding and uptake activity. Consistent with this, solid-phase immunoassays showed the p.(Cys46Gly) LDLR variant has decreased binding affinity for apolipoproteins. These results indicate the important role of Cys46 in LDL receptor activity and highlight the role of LR1 in LDLr activity modulation. This study reinforces the significance of in vitro functional characterization of LDL receptor activity in developing an accurate approach to FH genetic diagnosis. This is of particular importance because it enables clinicians to tailor personalized treatments for patients' mutation profile.

Increased intestinal cholesterol absorption in autosomal dominant hypercholesterolemia and no mutations in the low-density lipoprotein receptor or apolipoprotein B genes.

CONTEXT: Autosomal dominant hypercholesterolemia (ADH) is frequently caused by functional mutations in the low-density lipoprotein receptor (LDLR) or apolipoprotein B-100 (APOB) genes, but approximately 40% of ADH subjects disclose no such molecular defects, possibly pointing to alternative genetic mechanisms. OBJECTIVE: Our objective was to test the hypothesis that increased intestinal cholesterol absorption might play a role in the lipid abnormalities of subjects with ADH without identified genetic defects. DESIGN AND SETTING: This is a cross-sectional study of consecutive subjects with primary hyperlipidemia identified during an 18-month period in two lipid clinics. STUDY SUBJECTS: A total of 52 subjects with a clinical diagnosis of ADH were examined for molecular defects in LDLR and APOB. No APOB defects were found. Functional LDLR mutations occurred in 31 (60%) subjects, who received a diagnosis of familial hypercholesterolemia (FH). Those for whom no mutations could be identified were labeled as non-FH ADH. In addition, 38 subjects with familial combined hyperlipidemia (FCH) and 45 normolipidemic control subjects were studied. INTERVENTIONS: Interventions were diagnostic. MAIN OUTCOME MEASURES: Serum noncholesterol sterols were used as markers for the efficiency of intestinal cholesterol absorption. RESULTS: Adjusted campesterol to cholesterol ratios increased in the order non-FH ADH more than FH more than controls more than FCH, with mean values (95% confidence interval) in 10(2) mmol/mol cholesterol of 505 (424-600), 397 (345-458), 335 (294-382), and 284 (247-328), respectively. Thus, cholesterol absorption was lowest in FCH and highest in non-FH ADH. CONCLUSIONS: Increased intestinal cholesterol absorption may partially explain the high cholesterol levels of non-FH ADH subjects. Serum noncholesterol sterols are a useful tool for the differential diagnosis of genetic hypercholesterolemias, especially FCH and ADH unrelated to LDLR or APOB defects.

Hyperlipoproteinaemia(a) is a common cause of autosomal dominant hypercholesterolaemia.

UNLABELLED: Autosomal dominant hypercholesterolaemia (ADH) are a heterogeneous group of monogenic lipid disorders. The plasma level of lipoprotein(a) (Lp(a)) is a heritable trait associated with increased coronary heart disease (CHD) risk. OBJECTIVE: To evaluate the frequency of elevated Lp(a) as a cause of ADH and the characteristics of subjects with high Lp(a) (hyperLp(a)). MATERIAL AND METHODS: 200 healthy subjects and 933 unrelated Spanish subjects with a clinical diagnosis of ADH who were screened for low-density lipoprotein receptor (LDLR) and apolipoprotein B (APOB) gene mutations. Standard cardiovascular risk factors and blood lipid levels, including Lp(a), were evaluated. HyperLp(a) was defined as Lp(a) levels >or=95th centile of control values. RESULTS: Lp(a) was higher in 263 subjects without LDLR or APOB mutations (nonLDLR/nonAPOB group) than in 670 subjects with mutations (FH group): 40.0 mg/dl (interquartile range (IR) 15.0-89.0) versus 31.0 mg/dl (IR 11.0-73.7) respectively, p = 0.002. HyperLp(a) was present in 23% of ADH subjects (odds ratio (OR) 5.6 (95% CI, 2.9 to 10.7) versus controls) and 29% of nonLDLR/nonAPOB subjects (OR 7.7; 3.9 to 15.4). After adjusting for Lp(a), LDL cholesterol levels were <95th centile in 28 (10.6%) nonLDLR/nonAPOB subjects and in 9 (1.3%) FH subjects. Lp(a) levels were nonsignificantly higher in ADH subjects with early-onset CHD than in those without (43.5 mg/dl, (IR, 12.0-82.0) versus 31.7 mg/dl (11.8-76.5), respectively). CONCLUSIONS: HyperLp(a) is responsible for ADH in approximately 6% of nonLDLR/nonAPOB subjects. HyperLp(a) would not appear to be a risk factor for early-onset CHD in ADH, independently of whether genetic defects have or have not been demonstrated.

Bile acid synthesis precursors in subjects with genetic hypercholesterolemia negative for LDLR/APOB/PCSK9/APOE mutations. Association with lipids and carotid atherosclerosis.

Some oxysterols are precursors of bile acid synthesis and play an important role in cholesterol homeostasis. However, if they are involved in the pathogeny of genetic hypercholesterolemia has not been previously explored. We have studied non-cholesterol sterol markers of cholesterol synthesis (lanosterol and desmosterol) and oxysterols (7α-hydroxy-4-cholesten-3-one, 24S-hydroxycholesterol and 27-hydroxycholesterol) in 200 affected subjects with primary hypercholesterolemia of genetic origin, negative for mutations in LDLR, APOB, PCSK9 and APOE genes (non-FH GH) and 100 normolipemic controls. All studied oxysterols and cholesterol synthesis markers were significantly higher in affected subjects than controls (P<0.001). Ratios of oxysterols to total cholesterol were higher in non-FH GH than in controls, although only 24S-hydroxycholesterol showed statistical significance (P<0.001). Cholesterol synthesis markers had a positive correlation with BMI, triglycerides, cholesterol and apoB in control population. However, these correlations disappeared in non-FH GH with the exception of a weak positive correlation for non-HDL cholesterol and apoB. The same pattern was observed for oxysterols with high positive correlation in controls and absence of correlation for non-FH GH, except non-HDL cholesterol for 24S-hydroxycholesterol and 27-hydroxycholesterol and apoB for 27-hydroxycholesterol. All non-cholesterol sterols had positive correlation among them in patients and in controls. A total of 65 (32.5%) and 35 (17.5%) affected subjects presented values of oxysterols ratios to total cholesterol above the 95th percentile of the normal distribution (24S-hydroxycholesterol and 27-hydroxycholesterol, respectively). Those patients with the highest levels of 24S-hydroxycholesterol associated an increase in the carotid intima media thickness. These results suggest that bile acid metabolism is affected in some patients with primary hypercholesterolemia of genetic origin, negative for mutations in the candidate genes, and may confer a higher cardiovascular risk. Our results confirm that cholesterol synthesis overproduction is a primary defect in non-HF GH and suggest that subjects with non-FH GH show high levels of oxysterols in response to hepatic overproduction of cholesterol.

Identification of a novel mutation in exon 13 of the LDL receptor gene causing familial hypercholesterolemia in two Spanish families.

DNA from 30 unrelated Spanish patients with familial hypercholesterolemia (FH) was studied by single-strand conformation polymorphisms (SSCP)/heteroduplex analysis for mutation detection in exon 13 of low density lipoprotein (LDL) receptor gene. Two patients were found to have an abnormal pattern by heteroduplex analysis, and direct sequencing revealed a C to G substitution at nucleotide position 1965, that results in a Phe to Leu change in codon 634, F634L. We have developed a PCR based assay to detect this mutation in family members. We found three additional F634L mutation carriers, and all of them had high cholesterol levels. Haplotype analysis revealed that all F634L mutation carriers had the same allele determined by TaqI -, StuI +, AvaII +, NcoI -, suggesting the presence of a common ancestor. We report a novel mutation located in exon 13 of the LDL receptor gene that causes FH. We also demonstrate the importance of combining SSCP and heteroduplex analysis to improve mutation detection.

A common variant in the ABCA1 gene is associated with a lower risk for premature coronary heart disease in familial hypercholesterolaemia.

Familial hypercholesterolaemia (FH) is a common autosomal codominant hereditary disease caused by defects in the LDL receptor (LDLR) gene, and one of the most common characteristics of affected subjects is premature coronary heart disease (CHD). In heterozygous FH patients, the clinical expression of FH is highly variable in terms of the severity of hypercholesterolaemia and the age of onset and severity of CHD. Identification of mutations in the ATP binding cassette transporter 1 (ABCA1) gene in patients with Tangier disease, who exhibit reduced HDL cholesterol and apolipoprotein A1 concentrations and premature coronary atherosclerosis, has led us to hypothesise that ABCA1 could play a key role in the onset of premature CHD in FH. In order to know if the presence of the R219K variant in the ABCA1 gene could be a protective factor for premature CHD in FH, we have determined the presence of this genetic variant by amplification by PCR and restriction analysis in a group of 374 FH subjects, with and without premature CHD. The K allele of the R219K variant was significantly more frequent in FH subjects without premature CHD (0.32, 95% CI 0.27 to 0.37) than in FH subjects with premature CHD (0.25, 95% CI 0.21 to 0.29) (p<0.05), suggesting that the genetic variant R219K in ABCA1 could influence the development and progression of atherosclerosis in FH subjects. Moreover, the K allele of the R219K polymorphism seems to modify CHD risk without important modification of plasma HDL-C levels, and it appears to be more protective for smokers than non-smokers.

Circulating soluble low-density lipoprotein receptor-related protein 1 (sLRP1) concentration is associated with hypercholesterolemia: A new potential biomarker for atherosclerosis.

BACKGROUND: There is clinical interest in identifying novel lipid biomarkers for evaluating cardiovascular risk and targeting lipid-lowering treatment. Low-density lipoprotein receptor-related protein 1 (LRP1) plays a crucial role in the dysregulated cholesterol transfer from modified lipoproteins to human coronary vascular smooth muscle cells (hVSMCs), promoting hVSMC-derived foam cell formation. LRP1 has a soluble and circulating form (sLRP1) generated from LRP1. Cholesterol modulates the release of the soluble form of LRP1. Using in vitro, ex vivo and patient-based approaches, we tested the association between circulating sLRP1 concentrations and hypercholesterolemia and the potential of sLRP1 as a biomarker of atherosclerosis. METHODS AND RESULTS: Circulating sLRP1 concentrations were higher in severe hypercholesterolemia compared to moderate hypercholesterolemia or normocholesterolemia (Study 1). Circulating sLRP1 was significantly associated with established pro-atherogenic lipid parameters in two different hypercholesterolemic populations (Studies 2 and 3). sLRP1 concentrations decreased after statin treatment and increased after statin withdrawal (Study 3). In vitro experiments showed that native LDL, aggregated LDL and VLDL+IDL lipoproteins induced the release of sLRP1 from hVSMC. sLRP1 levels were increased in the conditioned medium of coronary atherosclerotic plaque areas extracted from patients compared to non-atherosclerotic areas of the same coronary artery and patient. Circulating sLRP1 concentrations were independently associated with the occurrence of carotid atherosclerosis in a hypercholesterolemic population (Study 2). The later association was higher than that observed for other classical or novel lipid parameters. CONCLUSIONS: Circulating sLRP1 is a new lipid-related parameter potentially useful as a biomarker for atherosclerosis.

Mutation analysis in 36 unrelated Spanish subjects with familial hypercholesterolemia: identification of 3 novel mutations in the LDL receptor gene.

We used the single strand conformation polymorphism (SSCP) method to investigate 36 apparently unrelated Spanish patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequences of the low density lipoprotein receptor (LDLR) gene. Nineteen aberrant SSCP patterns were found, and the underlying mutations were characterized by DNA sequencing. In addition, we tested all patients for the presence of mutations in the gene coding for apolipoprotein B (apo B). Five missense mutations (Q71E, S156L, E256K, N543H and T705I), four nonsense mutations (W(-18)X, E10X, Q133X and C255X), six frameshift mutations (211delG, 518delG, 1045delC, 2085del19, 2207insT and 2393del9) and five splicing mutations (313+1G->C, 1061-8T->C, 1845+1G->C, 2140+5G->A and 2390-1G->C) were identified in the LDLR gene. In total, we detected 20 mutations, 3 of which, designated 1045delC, 1845+1G->C and 2207insT, have not been previously described. Seven patients were found to carry two different mutations in the same allele: W(-18)X and E256K (one patient), Q71E and 313+1G->C (two patients), 1061-8T->C and T705I (two patients), 518delG and 2140+5G->A (one patient) and N543H and 2393del9 (one patient). As we expected, there is a broad spectrum of mutations in the LDLR gene, given the genetic heterogeneity of the Spanish population.

A double mutant [N543H+2393del9] allele in the LDL receptor gene in familial hypercholesterolemia: effect on plasma cholesterol levels and cardiovascular disease.

Familial hypercholesterolemia is a genetic disorder caused by mutations in the LDL receptor gene. During a survey of mutations of LDL receptor gene in Spanish FH patients we found two mutations in the same allele: a missense N543H mutation in exon 11 and a 9bp inframe deletion (2393del9) located in exon 17. This double mutant allele was founded in 10 out of 458 unrelated patients: one homozygous FH [N543H+2393del9] + [N543H+2393del9], one compound heterozygote [N543H+2393del9] + [W-18X+E256K] and 8 heterozygotes. Flow cytometric analysis showed a defective LDL binding (20% of normal value) and internalization (23%) in lymphocytes from the homozygous patient; furthermore, studies of mitogen-stimulated lymphocytes demonstrated that the ability of LDL to support cell proliferation was impaired. Unexpectedly, not all carriers of the double mutant allele develop hypercholesterolemia and, furthermore, cholesterol-lowering treatment of the homozygous patient resulted in a 58% LDL cholesterol reduction. In conclusion, the phenotypic expression in the homozygous and heterozygous patients presented here, as well as the LDL-receptor residual activity, allowed the classification of this mutation as mild extending the group of mild mutations found at homozygosity.