Familial Hypercholesterolemia Subjects Research Articles

Oxidative stress and antioxidant enzyme values in lymphomonocytes after an oral unsaturated fat load test in familial hypercholesterolemic subjects

Oxidative stress (OS) has been observed in conditions affecting the cardiovascular system. Familial hypercholesterolemia (FH) is associated with an increased risk of premature coronary heart disease. In the postprandial state, circulating lipids and lipoproteins can modulate OS status. Our aim was to study the response of lymphomonocyte OS status and reactive oxygen species by-products after an oral unsaturated fat load test (OFLT) in those with FH and to compare this response with that obtained in normolipidemic, normoglycemic subjects. We studied 12 patients with FH and 20 healthy controls. In both groups, lymphomonocyte, oxidized/reduced glutathione ratio, and malondialdehyde were determined at baseline and at 2, 4, 6, and 8 hours after an OFLT. Fasting urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine and isoprostane were measured using standard procedures. In both groups, oxidized/reduced glutathione ratio and malondialdehyde significantly decreased in the postprandial state after the OFLT. Both parameters were significantly higher in the FH group at baseline and during all the postprandial points, but the reduction from the baseline levels was significantly higher in the FH group than in the control group. Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine was significantly increased in the FH group compared with the healthy control group, indicating a higher fasting OS status. We conclude that subjects with FH exhibited OS levels that were higher than in controls before and after an OFLT, but the improvement in the OS status after the unsaturated fat load was significantly higher in subjects with FH.

Familial Hypercholesterolemia in the Danish General Population: Prevalence, Coronary Artery Disease, and Cholesterol-Lowering Medication

The diagnosis of familial hypercholesterolemia (FH) can be made using the Dutch Lipid Clinic Network criteria. This employs the personal and family history of premature coronary artery disease and hypercholesterolemia and the presence of a pathogenic mutation in the low-density lipoprotein receptor (LDLR) and apolipoprotein B (APOB) genes. We employed this tool to investigate the prevalence of FH and the associations between FH and coronary artery disease and cholesterol-lowering medication in the Copenhagen General Population Study. The study was of an unselected, community-based population comprising 69,016 participants. FH (definite/probable) was defined as a Dutch Lipid Clinic Network score higher than 5. Coronary artery disease was myocardial infarction or angina pectoris. The prevalence of FH was 0.73% (one in 137). Of participants with FH, 20% had an LDLR or APOB mutation. The prevalence of coronary artery disease among FH participants was 33%. Only 48% of subjects with FH admitted to taking cholesterol-lowering medication. The odds ratio for coronary artery disease off cholesterol-lowering medication was 13.2 (10.0-17.4) in definite/probable FH compared with non-FH subjects, after adjusting for age, gender, body mass index, hypertension, metabolic syndrome and diabetes, and smoking. The corresponding adjusted odds ratio for coronary artery disease in FH subjects on cholesterol-lowering medication was 10.3 (7.8-13.8). The prevalence of FH appears to be higher than commonly perceived in a general population of white Danish individuals, with at least half of affected subjects not receiving cholesterol-lowering medication. The very high risk of coronary artery disease irrespective of use of medication reflects the extent of underdiagnosis and undertreatment of FH in the community and primary care.

Post‐prandial remnant lipoprotein metabolism in autosomal recessive hypercholesterolaemia

Phenotype of autosomal recessive hypercholesterolaemia (ARH), a rare lipid disorder, is known to be milder than that of homozygous familial hypercholesterolaemia (FH) with LDL receptor gene mutation. However, few data exist regarding the functional differences in ARH and FH particularly in terms of remnant-like particles' (RLP) metabolism. Blood sampling was performed up to 6h after OFTT cream loading (50 g/body surface area) with 2-h intervals in a single ARH proband, four heterozygous FH patients with LDL receptor gene mutation and four normal controls. Plasma lipoprotein and RLP fraction were determined by HPLC system. The area under curve (AUC) of each lipoprotein including RLP fractions was evaluated. The AUC of TG, RLP cholesterol (RLP-C) and RLP triglyceride (RLP-TG) levels of heterozygous FH subjects was significantly higher than those of controls (466±71 mg/dL×h vs. 303±111 mg/dL×h, P<0·05; 35±7 mg/dL×h vs. 21±8 mg/dL×h, P<0·05; 124±57 mg/dL×h vs. 51±13 mg/dL×h, P<0·05, respectively). Under these conditions, those values of ARH were close to those of controls (310 mg/dL×h, 22 mg/dL×h, 23 mg/dL×h, respectively). These data demonstrate that unlike in FH, RLP clearance is preserved in ARH. The preservation of post-prandial RLP clearance may contribute to the mild phenotype of ARH compared with FH.

Aféresis de lipoproteínas de baja densidad en la hipercolesterolemia familiar resistente al tratamiento médico convencional intensivo

Background and objectiveThe aim of our study is to analyze the effect and security of low-density lipoproteins (LDL) apheresis in familial hypercholesterolemia (FH) subjects who did not response to conventional intensive optimized medical treatment. Patients and methodsSeven heterozygous FH subjects and one homozygous apoB familial defective were studied. All subjects were on secondary prevention with highest statins doses in association with other hypolipemiant drugs; the mean LDL-C reduction was 20%. All of them were treated with LDL apheresis (immunoabsorption) for a mean of 4.25 years. ResultsLDL apheresis resulted in a 68.3% decrease in LDL-C and 58.2% in apoB plasma values (P<.001). After an average of 3 years of follow-up, the cardiovascular events disappeared in 4 out of 5 symptomatic patients while in one patient the events were reduced in 75%. Four moderate side effects were reported in 820 apheresis procedures. ConclusionsLDL apheresis is a well-tolerated and safe treatment in FH patients who do not response to intensive conventional optimized medical treatment. The main limitation is its economical cost and low accessibility.

Detection of subclinical atherosclerosis in familial hypercholesterolemia using non-invasive imaging modalities

ObjectivesTo investigate the extent of subclinical atherosclerosis in asymptomatic familial hypercholesterolemia (FH) patients using non-invasive images techniques. Patients, methods and resultsThe atherosclerotic burden of 36 molecularly defined FH patients (18 males, 45.7±10.9 years) without evidence of cardiovascular disease receiving lipid-lowering treatment and 19 (47.8±11.3 years) controls was investigated. Descending thoracic aorta magnetic resonance imaging (MRI) was performed in a 1.5T equipment with T1 and T2 sequences to characterize atherosclerotic plaques and to measure aortic wall volumen. Carotid intima-media thickness (cIMT) and presence of plaques were measured using B-mode carotid ultrasound.Mean aortic wall volumen, cIMT and atherosclerotic plaques in aorta were significantly higher in FH cases (P<0.001). A significant correlation between aortic wall volume and cIMT was observed (P<0.01). Aortic MRI detected plaques in 94% and carotid ultrasound in 14% of cases. Lipid-rich plaques were observed only in FH cases (33%) and were associated with family history of premature coronary artery disease (P<0.05). ConclusionsAsymptomatic middle-aged FH patients have significantly higher atherosclerotic burden than controls. cIMT has shown a significant correlation with aortic wall volume and MRI allowed the detection of lipid-rich plaques in FH subjects that were associated with family history of premature coronary artery disease.

Cardio-Ankle Vascular Index in Heterozygous Familial Hypercholesterolemia

The cardio-ankle vascular index (CAVI) is a new non-invasive marker of arterial stiffness and atherosclerosis. The purpose of this study was to compare CAVI in patients with heterozygous familial hypercholesterolemia (FH) and in healthy controls. 82 FH subjects (27 males, 65 females), aged 53.7±13.6 years without clinical symptoms of cardiovascular diseases and 359 healthy controls (121 males, 238 females), aged 43.9±14.9 years, were examined. CAVI was measured using the system VaSera® 1500. CAVI in FH patients was significantly higher (8.0±1.4) than in healthy subjects (7.5±1.3) p = 0.002; however, age, sex and BMI adjusted CAVI did not differ significantly (p = 0.061) between the FH group (7.5, CI: 7.3; 7.7) and control group (7.7, CI: 7.6; 7.7). The study showed no significant difference in CAVI between heterozygous FH and healthy controls.

The removal from plasma of chylomicrons and remnants is reduced in heterozygous familial hypercholesterolemia subjects with identified LDL receptor mutations: Study with artificial emulsions

Chylomicron remnants bind to both their specific receptors (LRP) and to the LDL receptor (LDLR) in the liver. There is controversy whether disturbances of chylomicron metabolism occur in subjects with familial hypercholesterolemia (FH). The aim of this study was to evaluate whether there are defects on the removal from plasma of chylomicrons and their remnants in heterozygous FH patients with determined LDLR mutations. We studied 20 heterozygous FH patients (43.2±12 years old, 60% males) and 50 normolipidemic subjects matched for age and gender. FH subjects were not in use of LDL-lowering drugs for at least 6 weeks. The removal from plasma of chylomicrons and their remnants was measured by isotopic decay after venous injection of a chylomicron-like emulsion radiolabeled with 14C-cholesteryl ester (14C-CE) and 3H-triolein (3H-TO). These track respectively removal from plasma of chylomicrons and remnants and lipolysis. There was a significant reduction in the fractional catabolic rates (FCR in h−1) of 14C-CE in FH in comparison with normolipidemics: 0.048 (1.46.10−7; 0.57) vs. 0.71(0.049; 1.62), [median (25th–75th percentile)], p=0.003. No differences were found in FCR of 3H-TO between FH and controls, respectively 1.62 (1.02; 2.331) and 1.914 (1.34; 2.878), p=0.405. In conclusion heterozygous FH subjects had a significant decrease on the removal from plasma of chylomicrons and their remnants compared with normolipidemics.

LDL-apheresis depletes apoE-HDL and pre-β1-HDL in familial hypercholesterolemia: relevance to atheroprotection

Subnormal HDL-cholesterol (HDL-C) and apolipoprotein (apo)AI levels are characteristic of familial hypercholesterolemia (FH), reflecting perturbed intravascular metabolism with compositional anomalies in HDL particles, including apoE enrichment. Does LDL-apheresis, which reduces HDL-cholesterol, apoAI, and apoE by adsorption, induce selective changes in HDL subpopulations, with relevance to atheroprotection? Five HDL subpopulations were fractionated from pre- and post-LDL-apheresis plasmas of normotriglyceridemic FH subjects (n = 11) on regular LDL-apheresis (>2 years). Apheresis lowered both plasma apoE (-62%) and apoAI (-16%) levels, with preferential, genotype-independent reduction in apoE. The mass ratio of HDL2:HDL3 was lowered from ~1:1 to 0.72:1 by apheresis, reflecting selective removal of HDL2 mass (80% of total HDL adsorbed). Pre-LDL-apheresis, HDL2 subpopulations were markedly enriched in apoE, consistent with ~1 copy of apoE per 4 HDL particles. Large amounts (50-66%) of apoE-HDL were removed by apheresis, preferentially in the HDL2b subfraction (-50%); minor absolute amounts of apoE-HDL were removed from HDL3 subfractions. Furthermore, pre-β1-HDL particle levels were subnormal following removal (-53%) upon apheresis, suggesting that cellular cholesterol efflux may be defective in the immediate postapheresis period. In LDL-receptor (LDL-R) deficiency, LDL-apheresis may enhance flux through the reverse cholesterol transport pathway and equally attenuate potential biglycan-mediated deposition of apoE-HDL in the arterial matrix.

Abstract 9712: Lack of Association Between Common p.T111I Variant in the Endothelial Lipase Gene and the Risk for Coronary Artery Disease in Familial Hypercholesterolemia: Insights From the Modulation of Phospholipids in the HDL Particles

Background: endothelial lipase (EL) is a major determinant of high-density lipoprotein cholesterol (HDL-C) metabolism through the lipolysis of the phospholipids (PL) in the HDL particles (HDL-PL). Several studies indicated that common p.T111I variant in EL gene (LIPG: rs2000813) is negatively associated with the risk of coronary artery disease (CAD) independent of HDL-C levels. However, few data exist regarding the influence of this variant on the content of HDL-PL in terms of the association with CAD, especially in familial hypercholesterolemia (FH) subjects. The aim of our study is to elucidate the clinical impact of common p.T111I variant in the EL gene on the content of HDL-PL, and whether this variant could be useful predictor of the risk of CAD in FH subjects, which always exhibit low HDL-C levels. Methods: The p.T111I variant was genotyped in our heterozygous FH subjects (n=80, male=56, mean age=64±18yr, mean TC=340±64mg/dl). All those subjects' plasma lipoproteins were investigated by ultracentrifugation method. Overall HDL-C level, HDL-PL level, and the existence of CAD were investigated for each group (TT group vs. TI and II group). Results: The minor allele frequency in the combined pools of subjects was 0.25. There was no significant association between this variant with overall HDL-C level (42±14 vs. 43±17mg/dl, p=NS) nor with HDL-PL level (86±24 vs. 88±27mg/dl, p=NS). In addition, multiple logistic regression analysis revealed no association between this variant and CAD (odds ratio 0.96, 95%CI:0.88-1.04, p=NS) although significant associations between several coronary risk factors and CAD were observed (figure). Conclusions: These data suggest that common p.T111I variant in LIPG neither plays a role in determining HDL-C as well as HDL-PL levels nor is a useful predictor of the risk of CAD in genetically determined heterozygous FH subjects.

Abstract 8252: Altered Metabolism of Post-Prandial Remnant Lipoprotein Fractions in Autosomal Recessive Hypercholesterolemia

Introduction: Autosomal recessive hypercholesterolemia (ARH) associated with mutations in LDLR adaptor protein 1 (LDLRAP1) gene is extremely rare disorder. Although this defect seems to be a phenocopy of homozygous familial hypercholesterolemia (FH), clinical phenotype of ARH seems less severe, more responsive to statins, the cause of which may be derived from the difference of remnant-like particles (RLP) clearance. Hypothesis: We assessed the hypothesis that the ability of RLP clearance of ARH subject was different from that of FH subject. Methods: OFTT cream (Jomo Shokuhin, Takasaki, Japan) 50 g was given per body surface area (squared meter), blood sampling was performed at 2 hour intervals up to 6 hour. Plasma lipoprotein and RLP fraction were determined by HPLC system in one ARH subject (68yr, LDL-C=286mg/dl), 4 heterozygous FH subjects (58±17yr, LDL-C=241±26mg/dl), and 4 controls (62±17yr, LDL-C=88±6mg/dl). All male subjects were selected to have apolipoprotein E2/E3 phenotype without diabetes. None of the subjects took medication known to affect plasma lipids at the time of this study for at least 4 weeks. The area under curve (AUC) of TG, RLP-TG, and RLP-cholesterol levels were evaluated. Results: After oral fat load, the AUC of TG, RLP-TG, and RLP-cholesterol levels of heterozygous FH subjects were significantly increased compared to those of controls (441±87 mg/dl*hour vs 316±133 mg/dl*hour, p&lt;0.05, 126±60 mg/dl*hour vs 49±11 mg/dl*hour, p&lt;0.05, 34±8 mg/dl*hour vs 23±11 mg/dl*hour, p&lt;0.05, respectively). In contrast to these results, the AUC of TG, RLP-TG, and RLP-cholesterol levels of ARH were within the nomal range (310mg/dl*hour, 23mg/dl*hour, 22mg/dl*hour, respectively). Conclusions: These results suggest that the lack of LDLRAP1 modulates RLP metabolism, activating an alternate pathway which can remove remnant fraction paradoxically. This preferred pathway should contribute to the greater responsiveness to statins. Our results will provide new insights into the lipoprotein metabolism of ARH and the novel pharmacological target for LDLRAP1.

MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins

Circulating microRNAs (miRNA) are relatively stable in plasma and are a new class of disease biomarkers. Here we present evidence that high-density lipoprotein (HDL) transports endogenous miRNAs and delivers them to recipient cells with functional targeting capabilities. Cellular export of miRNAs to HDL was demonstrated to be regulated by neutral sphingomyelinase. Reconstituted HDL injected into mice retrieved distinct miRNA profiles from normal and atherogenic models. HDL delivery of both exogenous and endogenous miRNAs resulted in the direct targeting of mRNA reporters. Furthermore, HDL-mediated delivery of miRNAs to recipient cells was demonstrated to be scavenger receptor BI-dependent. The human HDL-miRNA profile from normal subjects is significantly different than familial hypercholesterolemia subjects. Notably, HDL-miRNA from atherosclerotic subjects induced differential gene expression, with significant loss of conserved mRNA targets in cultured hepatocytes. Collectively, these observations suggest that HDL participates in a mechanism of intercellular communication involving the transport and delivery of miRNAs.

Molecular Characterization of Iranian Patients with Possible Familial Hypercholesterolemia

Familial hypercholesterolemia (FH) is an autosomal dominant disorder of lipoprotein metabolism caused mainly by mutations in the low-density lipoprotein receptor (LDLR) and apolipoprotein B 100 (APOB) genes. Until now, the molecular basis of FH has been demonstrated in detail in many populations, but there is still very limited Molecular data concerning FH in Iran. The aim of this study was to characterize the LDLR and APOB gene mutations in an Iranian population. A total of 30 non-related Iranian possible FH subjects were studied. Diagnosis of FH was based on the Dutch Lipid Clinic Network diagnostic criteria. All samples were initially tested for three common APOB gene mutations including R3500Q, R3500W and R3531C using PCR-RFLP assay. Subsequently, promoter and coding region of the LDLR gene was screened by PCR-SSCP analysis and positive results were confirmed by DNA sequencing. Four previously reported polymorphisms 1413G>A, 1725C>T, 1773T>C and 2140+5G>A were found in ~17% (5/30) of population studied. Moreover, no variation was found in APOB gene. Our data indicated that LDLR and APOB gene mutations have not contribution to possible FH in Iranian population studied here. However, we examined three common APOB mutations and LDLR in only 30 patients, and to determine the role of these genes in developing FH in Iran, more FH samples and populations needed to be investigated for the mutations of the related genes.

Clinical characteristics and evaluation of LDL-cholesterol treatment of the Spanish Familial Hypercholesterolemia Longitudinal Cohort Study (SAFEHEART)

AimFamilial hypercholesterolemia (FH) patients are at high risk for premature coronary heart disease (CHD). Despite the use of statins, most patients do not achieve an optimal LDL-cholesterol goal. The aims of this study are to describe baseline characteristics and to evaluate Lipid Lowering Therapy (LLT) in FH patients recruited in SAFEHEART.Methods and ResultsA cross-sectional analysis of cases recruited in the Spanish FH cohort at inclusion was performed. Demographic, lifestyle, medical and therapeutic data were collected by specific surveys. Blood samples for lipid profile and DNA were obtained. Genetic test for FH was performed through DNA-microarray. Data from 1852 subjects (47.5% males) over 19 years old were analyzed: 1262 (68.1%, mean age 45.6 years) had genetic diagnosis of FH and 590 (31.9%, mean age 41.3 years) were non-FH. Cardiovascular disease was present in 14% of FH and in 3.2% of non-FH subjects (P < 0.001), and was significantly higher in patients carrying a null mutation compared with those carrying a defective mutation (14.87% vs. 10.6%, respectively, P < 0.05). Prevalence of current smokers was 28.4% in FH subjects. Most FH cases were receiving LLT (84%). Although 51.5% were receiving treatment expected to reduce LDL-c levels at least 50%, only 13.6% were on maximum statin dose combined with ezetimibe. Mean LDL-c level in treated FH cases was 186.5 mg/dl (SD: 65.6) and only 3.4% of patients reached and LDL-c under 100 mg/dl. The best predictor for LDL-c goal attainment was the use of combined therapy with statin and ezetimibe.ConclusionAlthough most of this high risk population is receiving LLT, prevalence of cardiovascular disease and LDL-c levels are still high and far from the optimum LDL-c therapeutic goal. However, LDL-c levels could be reduced by using more intensive LLT such as combined therapy with maximum statin dose and ezetimibe.

Myeloperoxidase modulation by LDL apheresis in Familial Hypercholesterolemia

BackgroundMyeloperoxidase (MPO) is a marker of plaque vulnerability and a mechanistic bridge between inflammation and cardiovascular disease, and thus is a suitable target for therapeutic strategy against cardiovascular disease.MethodsSince hypercholesterolemia is associated with atherosclerosis and inflammation, we tested whether MPO serum levels were up-regulated in Familial Hypercholesterolemia (FH) and whether acute reduction of total cholesterol (TC) would also reduce MPO concentration. FH subjects undergoing LDL-apheresis (LDL-A) treatment are a paradigmatic clinical model where TC rapidly plunges from extremely high to extremely low levels after selective LDL removal, and then spontaneously rebounds to baseline conditions. This clinical setting allows multiple intra-patient observations at different plasma TC concentrations. We measured MPO levels in serum by ELISA tests, and in peripheral leukocytes by immunofluorescence, to learn whether they were affected by the changes in TC levels. Serum MPO was measured before and serially up to the 14th day following LDL-A.ResultsIn both serum and peripheral leukocytes, MPO concentrations were i) higher than in sex- and age-matched healthy controls (p < 0.01); ii) decreased with TC reduction; iii) parallel with TC time course; iv) correlated with plasma TC. At regression analysis, plasma TC was the only variable considered that influenced MPO serum levels (β 0.022 ± 0.010, p < 0.0001).ConclusionsIn FH the MPO serum levels were modulated through changes in the TC concentrations carried out by LDL-A. Further study is needed to determine whether reduced MPO levels obtained by LDL-A could have any therapeutic impact.

Evaluation of subclinical atherosclerosis by computed tomography coronary angiography and its association with risk factors in familial hypercholesterolemia

Background Increasing age and cholesterol levels, male gender, and family history of early coronary heart disease (CHD) are associated with early onset of CHD in familial hypercholesterolemia (FH). Objective Assess subclinical atherosclerosis by computed tomography coronary angiography (CTCA) and its association with clinical and laboratorial parameters in asymptomatic FH subjects. Methods 102 FH subjects (36% male, 45 ± 13 years, LDL-c 280 ± 54 mg/dL) and 35 controls (40% male, 46 ± 12 years, LDL-c 103 ±18 mg/dL) were submitted to CTCA. Plaques were divided into calcified, mixed and non-calcified; luminal stenosis was characterized as >50% obstruction. Results FH had a greater atherosclerotic burden represented by higher number of patients with: plaques (48% vs. 14%, p = 0.0005), stenosis (19% vs. 3%, p = 0.015), segments with plaques (2.05 ± 2.85 vs.0.43 ± 1.33, p = 0.0016) and calcium scores (55 ± 129 vs. 38 ± 140, p = 0.0028). After multivariate analysis, determinants of plaque presence were increasing age (OR = 2.06, for age change of 10 years, CI95%: 1.38–3.07, p < 0.001) and total cholesterol (OR = 1.86, for cholesterol change by 1 standard deviation, CI95%: 1.09–3.15, p = 0.027). Coronary calcium score was associated with the presence of stenosis (OR = 1.54; CI95%: 1.27–1.86, p < 0.001, for doubling the calcium score). Male gender was directly associated with the presence of non-calcified plaques (OR: 15.45, CI95% 1.72–138.23, p = 0.014) and inversely with calcified plaques (OR = 0.21, CI95%: 0.05–0.84, p = 0.027). Family history of early CHD was associated with the presence of mixed plaques (OR = 4.90, CI95%: 1.32–18.21, p = 0.018). Conclusions Patients with FH had an increased burden of coronary atherosclerosis by CTCA. The burden of atherosclerosis and individual plaque subtypes differed with the presence of other associated risk factors, with age and cholesterol being most important. A coronary calcium score of zero ruled out obstructive disease in this higher risk population.

Familial hypercholesterolemia and triglyceride metabolism

Familial hypercholesterolemia (FH) is a common autosomal disorder associated with hypercholesterolemia which usually results from a mutation in the coding region of the low density lipoprotein (LDL) receptor (R) activity. Only 20% of untreated heterozygote (h) FH men reach 70 years of age. Therefore, the diagnosis of hFH is a better predictor of coronary heart disease than risk-based algorithms. Fasting and postprandial hypertriglyceridemia are also considered as risk factors for atherosclerosis. The plasma triglycerides (TG)s are formed from two major sources; intestinally-derived chylomicrons and hepatically-derived very low density lipoproteins (VLDL). Potentially, atherogenic remnants of TG-rich lipoproteins accumulate in the postprandial state. In addition, TG-rich lipoproteins may promote the formation of atherogenic small dense LDL. In FH subjects, lipoprotein metabolism seems to be impaired and may contribute to premature atherosclerosis. This was documented in many studies in which mice lacking LDLR present hypercholesterolemia, increased plasma TG-rich lipoprotein remnants and develop premature spontaneous atherosclerosis. In this review, we focus on the current knowledge regarding TG metabolism on a selected clinically condition such as FH. Variation in clinical characteristics has been described between studies which may occur due to dissimilarity in the molecular defect of FH. Additionally, the relationship between TG levels in FH subjects and the development of atherosclerosis, as well as the appropriate treatment for these patients is analysed.

Long-term LDL-c lowering in heterozygous familial hypercholesterolemia normalizes carotid intima-media thickness

Objective We investigated the effectiveness of statins in daily practice in reducing the arterial wall thicknesses by comparing the carotid intima-media thickness (cIMT) between statin-treated familial hypercholesterolemia (FH) patients and their unaffected spouses. Methods FH subjects treated with LDL-c lowering medication for at least 5 years and their unaffected spouses were included in this observational study. Clinical data and carotid intima-media thickness (cIMT) as surrogate marker for atherosclerosis were acquired. Results In total 40 FH patients, age 48.4 ± 4.2 years, and their 40 unaffected spouses, age 47.4 ± 3.9 years, were included. Pre-treatment total cholesterol levels of FH patients were on average 9.3 ± 2.0 mmol/L. Treated FH patients and unaffected spouses exhibited similar LDL-c (3.8 ± 1.5 vs. 3.5 ± 1.1 mmol/L; p = 0.25) and total cholesterol levels (5.8 ± 1.6 vs. 5.6 ± 1.1 mmol/L; p = 0.56). Also, in a multivariate model cIMT adjusted for age and sex did not differ between affecteds and spouses (95% CI: −0.032 to 0.092 mm; p = 0.34). Conclusion Long-term statin treatment normalizes cIMT in severe FH patients and therefore it is likely that the extreme risk of cardiovascular disease in FH patients is significantly reduced by this therapy.

A new method for measurement of total plasma PCSK9: clinical applications

The proprotein convertase subtilisin kexin-9 (PCSK9) circulates in plasma as mature and furin-cleaved forms. A polyclonal antibody against human PCSK9 was used to develop an ELISA that measures total plasma PCSK9 rather than only the mature form. A cross-sectional study evaluated plasma levels in normal (n = 254) and hypercholesterolemic (n = 200) subjects treated or untreated with statins or statin plus ezetimibe. In controls, mean plasma PCSK9 (89.5 +/- 31.9 ng/ml) correlated positively with age, total cholesterol, LDL-cholesterol (LDL-C), triglycerides, and fasting glucose. Sequencing PCSK9 from individuals at the extremes of the normal PCSK9 distribution identified a new loss-of-function R434W variant associated with lower levels of circulating PCSK9 and LDL-C. In hypercholesterolemic subjects, PCSK9 levels were higher than in controls (99.3 +/- 31.7 ng/ml, P < 0.04) and increased in proportion to the statin dose, combined or not with ezetimibe. In treated patients (n = 139), those with familial hypercholesterolemia (FH; due to LDL receptor gene mutations) had higher PCSK9 values than non-FH (147.01 +/- 42.5 vs. 127.2 +/- 40.8 ng/ml, P < 0.005), but LDL-C reduction correlated positively with achieved plasma PCSK9 levels to a similar extent in both subsets (r = 0.316, P < 0.02 in FH and r = 0.275, P < 0.009 in non-FH). The detection of circulating PCSK9 in both FH and non-FH subjects means that this assay could be used to monitor response to therapy in a wide range of patients.

Genetics and kinetics of familial hypercholesterolemia, with the special focus on FH- Marburg p.W556R

Objective Familial hypercholesterolemia (FH) is an autosomal dominant inherited disorder, caused by mutations in the low density lipoprotein receptor (LDLR) gene. FH is characterized by elevated plasma LDL cholesterol, premature atherosclerosis and high risk of premature myocardial infarction. Extended work has been done to understand both, the primary genetic defect as well as the in vivo kinetic consequences of this disease. Both approaches, genetics and kinetics, are challenging but also fruitful approaches for a better understanding of this devastating disease. For this we reviewed the recent literature and used our in vitro and in vivo data on one of the most frequently occurring types of FH, the FH Marburg p.W556R. Methods To identify the primary genetic defect of the FH Marburg we used denaturing gradient gel electrophoresis (DGGE) mutation analysis. In vivo kinetic studies were performed in a heterozygote FH Marburg subject and in 5 healthy control subjects utilizing a stable isotope tracer kinetic approach with 3D-leucine. Results DGGE screening of the LDLR gene identified a tryptophan (W) to arginine (R) substitution at residue 556 (p.W556R) in the fifth conserved YWTD repeat of the LDLR-β-propeller in FH Marburg. In vivo kinetic studies in a heterozygote FH subject for FH Marburg and in 5 healthy control subjects demonstrated a severe decrease in LDL FCR and a mild increase of LDL PR in FH compared to healthy controls. Conclusions The LDLR mutation p.W556R is a frequent and severe defect for FH. This defect has a major influence on the in vivo lipoprotein kinetics and lipid levels. In a heterozygote FH patient we found a dual defect for the increase in LDL cholesterol, namely a decrease in the fractional catabolic rate (FCR) of LDL but also an increase in LDL production rate (PR). By this a well defined, single genetic defect may have a series of different in vivo metabolic consequences which could be used for potential therapeutic approaches to this disease.

Development of a high-resolution melting method for mutation detection in familial hypercholesterolaemia patients

Current screening methods, such as single strand conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (dHPLC) that are used for detecting mutations in familial hypercholesterolaemia (FH) subjects are time consuming, costly and only 80-90% sensitive. Here we have tested high-resolution melt (HRM) analysis for mutation detection using the Rotor-Gene(6000) realtime rotary analyser. Methods and subjects Polymerase chain reaction and melt conditions (HRM) for 23 fragments of the LDL-receptor gene, a region of exon 26 in the APOB gene (including p.R3527Q) and exon 7 of the PCSK9 gene (including p.D374Y) were optimized. Two double stranded DNA saturating dyes, LC-Green and Syto9, were compared for sensitivity. Eighty-two samples with known mutations were used as positive controls. Twenty-eight Greek FH heterozygous patients and two homozygous patients from the UK and Croatia were screened. HRM was able to identify all the positive control mutations tested, with similar results with either dye. Eight different variations were found in 17 of the 28 Greek FH patients for an overall detection rate of 61%: c.41delT (1), p.W165X (1), p.C173R (3), p.S286R (2), p.V429M (4), p.G549D (4), p.V613I (1), and a previously unreported mutation p.F694V (1) which is predicted to be FH-causing by functional algorithms. Mutations were found in both the homozygous patients; p.Q92X (Croatia) and p.Y489C (UK); both patients were homozygous for their respective mutations. HRM is a sensitive, robust technique that could significantly reduce the time and cost of screening for mutations in a clinical setting.