Low-density Lipoprotein Receptor Alleles Research Articles

IPSC-Derived Endothelial Cells Reveal LDLR Dysfunction and Dysregulated Gene Expression Profiles in Familial Hypercholesterolemia.

Defects in the low-density lipoprotein receptor (LDLR) are associated with familial hypercholesterolemia (FH), manifested by atherosclerosis and cardiovascular disease. LDLR deficiency in hepatocytes leads to elevated blood cholesterol levels, which damage vascular cells, especially endothelial cells, through oxidative stress and inflammation. However, the distinctions between endothelial cells from individuals with normal and defective LDLR are not yet fully understood. In this study, we obtained and examined endothelial derivatives of induced pluripotent stem cells (iPSCs) generated previously from conditionally healthy donors and compound heterozygous FH patients carrying pathogenic LDLR alleles. In normal iPSC-derived endothelial cells (iPSC-ECs), we detected the LDLR protein predominantly in its mature form, whereas iPSC-ECs from FH patients have reduced levels of mature LDLR and show abolished low-density lipoprotein uptake. RNA-seq of mutant LDLR iPSC-ECs revealed a unique transcriptome profile with downregulated genes related to monocarboxylic acid transport, exocytosis, and cell adhesion, whereas upregulated signaling pathways were involved in cell secretion and leukocyte activation. Overall, these findings suggest that LDLR defects increase the susceptibility of endothelial cells to inflammation and oxidative stress. In combination with elevated extrinsic cholesterol levels, this may result in accelerated endothelial dysfunction, contributing to early progression of atherosclerosis and other cardiovascular pathologies associated with FH.

Association of LDLR rs1433099 with the Risk of NAFLD and CVD in Chinese Han Population.

Background and AimsRecent genome-wide association studies have shown that low-density lipoprotein receptor (LDLR) rs1433099 polymorphism is associated with cardiovascular disease (CVD) risk in many countries. However, the association of LDLR rs1433099 with CVD in China has not been reported yet. There are no studies on LDLR rs1433099 and non-alcoholic fatty liver disease (NAFLD) as well. The purpose of this study was to investigate whether LDLR rs1433099 is related to CVD or NAFLD in the Chinese population.MethodsLDLR rs1433099 polymorphism was genotyped in 507 individuals, including 140 healthy controls, 79 NAFLD patients, 185 CVD patients, and 103 patients with NAFLD combined with CVD. The expression of LDLR was tested by the sequence detection system, and clinical parameters were assessed by biochemical tests and physical examination.ResultsThe genotype distribution of LDLR rs1433099 was not statistically different among the NAFLD group, the CVD group, the combined group, and the healthy control group (p>0.05). There was no significant correlation of LDLR rs1433099 genotypic distribution or allele frequency and the risk of NAFLD, CVD or NAFLD combined with CVD (p>0.05). In the CVD group, T allele carriers had higher alkaline phosphatase and gamma-glutamyl transpeptidase than non-carriers (p<0.05).ConclusionsOur study demonstrated that the LDLR rs1433099 polymorphism is not a risk factor of NAFLD. The LDLR rs1433099 polymorphism may increase the risk of CVD through a mechanism involving alkaline phosphatase and gamma-glutamyl transpeptidase.

CRISPR Correction of a Homozygous Low-Density Lipoprotein Receptor Mutation in Familial Hypercholesterolemia Induced Pluripotent Stem Cells.

Familial hypercholesterolemia (FH) is a hereditary disease primarily due to mutations in the low‐density lipoprotein receptor (LDLR) that lead to elevated cholesterol and premature development of cardiovascular disease. Homozygous FH patients (HoFH) with two dysfunctional LDLR alleles are not as successfully treated with standard hypercholesterol therapies, and more aggressive therapeutic approaches to control cholesterol levels must be considered. Liver transplant can resolve HoFH, and hepatocyte transplantation has shown promising results in animals and humans. However, demand for donated livers and high‐quality hepatocytes overwhelm the supply. Human pluripotent stem cells can differentiate to hepatocyte‐like cells (HLCs) with the potential for experimental and clinical use. To be of future clinical use as autologous cells, LDLR genetic mutations in derived FH‐HLCs need to be corrected. Genome editing technology clustered‐regularly‐interspaced‐short‐palindromic‐repeats/CRISPR‐associated 9 (CRISPR/Cas9) can repair pathologic genetic mutations in human induced pluripotent stem cells. Conclusion: We used CRISPR/Cas9 genome editing to permanently correct a 3‐base pair homozygous deletion in LDLR exon 4 of patient‐derived HoFH induced pluripotent stem cells. The genetic correction restored LDLR‐mediated endocytosis in FH‐HLCs and demonstrates the proof‐of‐principle that CRISPR‐mediated genetic modification can be successfully used to normalize HoFH cholesterol metabolism deficiency at the cellular level. (Hepatology Communications 2017;1:886–898)

Contribution of mutations in low density lipoprotein receptor (LDLR) and lipoprotein lipase (LPL) genes to familial combined hyperlipidemia (FCHL): A reappraisal by using a resequencing approach

BackgroundDefective low-density lipoprotein receptor (LDLR) and lipoprotein lipase (LPL) alleles have been implicated in familial combined hyperlipidemia (FCHL). However, their contribution might have been influenced by diagnostic criteria. This study was aimed to reassess the frequency of rare and common variants in LDLR and LPL in FCHL individuals classified with stringent criteria. MethodsLDLR and LPL were resequenced in 208 FHCL and 171 controls. Variants were classified as loss- (LOF) or gain-of-function (GOF) based upon in silico prediction, familial segregation and available functional data. ResultsEight LOF variants were detected in LDLR, 6 of which were missense and 2 were predicted to disrupt normal splicing; all were present at heterozygous state. They were found in 10 FCHL but not in controls, thus indicating that 4.8% of FCHL individuals should be reclassified as FH. LDL-C (positive) and BMI (negative) were the strongest predictors of LDLR mutations with LDL-C 181 mg/dl being the best threshold for diagnosing the presence of dysfunctional LDLR alleles. The cumulative prevalence of definite LPL defective alleles (1 rare and 2 common heterozygous missense variants) was comparable between FCHL and controls (10.1% vs. 10.5%). Conversely, the LPL GOF variant p.Ser474* showed a lower frequency in FCHL than in controls (13.5% vs. 24.0%, p = 0.008). Overall, LOF LPL variants did not show a TG-modulating effect. ConclusionsOur findings indicate that, in well characterized FCHL individuals, variants in LDLR and LPL provide a small contribution to this dyslipidemia, thus limiting the need for such genetic testing.

Systematic cell-based phenotyping of missense alleles empowers rare variant association studies: a case for LDLR and myocardial infarction.

A fundamental challenge to contemporary genetics is to distinguish rare missense alleles that disrupt protein functions from the majority of alleles neutral on protein activities. High-throughput experimental tools to securely discriminate between disruptive and non-disruptive missense alleles are currently missing. Here we establish a scalable cell-based strategy to profile the biological effects and likely disease relevance of rare missense variants in vitro. We apply this strategy to systematically characterize missense alleles in the low-density lipoprotein receptor (LDLR) gene identified through exome sequencing of 3,235 individuals and exome-chip profiling of 39,186 individuals. Our strategy reliably identifies disruptive missense alleles, and disruptive-allele carriers have higher plasma LDL-cholesterol (LDL-C). Importantly, considering experimental data refined the risk of rare LDLR allele carriers from 4.5- to 25.3-fold for high LDL-C, and from 2.1- to 20-fold for early-onset myocardial infarction. Our study generates proof-of-concept that systematic functional variant profiling may empower rare variant-association studies by orders of magnitude.

Exome sequencing identifies rare LDLR and APOA5 alleles conferring risk for myocardial infarction.

SummaryMyocardial infarction (MI), a leading cause of death around the world, displays a complex pattern of inheritance1,2. When MI occurs early in life, the role of inheritance is substantially greater1. Previously, rare mutations in low-density lipoprotein (LDL) genes have been shown to contribute to MI risk in individual families3–8 whereas common variants at more than 45 loci have been associated with MI risk in the population9–15. Here, we evaluate the contribution of rare mutations to MI risk in the population. We sequenced the protein-coding regions of 9,793 genomes from patients with MI at an early age (≤50 years in males and ≤60 years in females) along with MI-free controls. We identified two genes where rare coding-sequence mutations were more frequent in cases versus controls at exome-wide significance. At low-density lipoprotein receptor (LDLR), carriers of rare, damaging mutations (3.1% of cases versus 1.3% of controls) were at 2.4-fold increased risk for MI; carriers of null alleles at LDLR were at even higher risk (13-fold difference). This sequence-based estimate of the proportion of early MI cases due to LDLR mutations is remarkably similar to an estimate made more than 40 years ago using total cholesterol16. At apolipoprotein A-V (APOA5), carriers of rare nonsynonymous mutations (1.4% of cases versus 0.6% of controls) were at 2.2-fold increased risk for MI. When compared with non-carriers, LDLR mutation carriers had higher plasma LDL cholesterol whereas APOA5 mutation carriers had higher plasma triglycerides. Recent evidence has connected MI risk with coding sequence mutations at two genes functionally related to APOA5, namely lipoprotein lipase15,17 and apolipoprotein C318,19. When combined, these observations suggest that, beyond LDL cholesterol, disordered metabolism of triglyceride-rich lipoproteins contributes to MI risk.

Select Articles Published on the Topic of Coronary Heart Disease in 2013

Select Articles Published on the Topic of Coronary Heart Disease in 2013

Circulation: Clinical Summaries

<i>Circulation:</i> Clinical Summaries

Reduction in Mortality in Subjects With Homozygous Familial Hypercholesterolemia Associated With Advances in Lipid-Lowering Therapy

Homozygous familial hypercholesterolemia is an inherited disorder caused by mutations in both low-density lipoprotein receptor alleles, which results in extremely elevated plasma low-density lipoprotein cholesterol concentrations and very early morbidity and mortality due to cardiovascular disease. To evaluate the impact of advances in lipid-lowering (predominantly statin) therapy on cardiovascular disease morbidity and mortality in a large cohort of patients with homozygous familial hypercholesterolemia, the records of 149 patients (81 females, 68 males) from 2 specialized lipid clinics in South Africa were evaluated retrospectively. Homozygous familial hypercholesterolemia was diagnosed by confirmation of mutations in genes affecting low-density lipoprotein cholesterol or by clinical criteria. A Cox proportional hazard model with time-varying exposure was used to estimate the risk of death and major adverse cardiovascular events among statin-treated patients compared with statin-naive patients. The hazard ratio for benefit from lipid therapy, calculated with the Cox proportional hazards model for the end point of death, was 0.34 (95% confidence interval 0.14-0.86; P=0.02), and for the end point of major adverse cardiovascular events, it was 0.49 (95% confidence interval 0.22-1.07; P=0.07). This occurred despite a mean reduction in low-density lipoprotein cholesterol of only 26.4% (from 15.9±3.9 to 11.7±3.4 mmol/L; P<0.0001) with lipid-lowering therapy. Lipid-lowering therapy is associated with delayed cardiovascular events and prolonged survival in patients with homozygous familial hypercholesterolemia.

Protein kinase C activation stabilizes LDL receptor mRNA via the JNK pathway in HepG2 cells

LDL is the most abundant cholesterol transport vehicle in plasma and a major prognostic indicator of atherosclerosis. Hepatic LDL receptors limit circulating LDL levels, since cholesterol internalized by the liver can be excreted. As such, mechanisms regulating LDL receptor expression in liver cells are appealing targets for cholesterol-lowering therapeutic strategies. Activation of HepG2 cells with phorbol esters enhances LDL receptor mRNA levels through transcriptional and posttranscriptional mechanisms. Here, we show that 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced stabilization of receptor mRNA requires the activity of protein kinase C and is accompanied by activation of the major mitogen activated protein kinase pathways. Inhibitor studies demonstrated that receptor mRNA stabilization is independent of the extracellular signal-regulated kinase or p38(MAPK), but requires activation of the c-Jun N-terminal kinase (JNK). An essential role for JNK in stabilizing receptor mRNA was further confirmed through small interfering RNA (siRNA) experiments and by activating JNK through two protein kinase C-independent mechanisms. Finally, prolonged JNK activation increased steady-state levels of receptor mRNA and protein, and significantly enhanced cellular LDL-binding activity. These data suggest that JNK may play an important role in posttranscriptional control of LDL receptor expression, thus constituting a novel mechanism to enhance plasma LDL clearance by liver cells.

LDLR promoter variant and exon 14 mutation on the same chromosome are associated with an unusually severe FH phenotype and treatment resistance

Familial hypercholesterolemia (FH) is the most common form of autosomal-dominant hypercholesterolemia, and is caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Heterozygous FH is characterized by elevated low-density lipoprotein (LDL) cholesterol and early-onset cardiovascular disease, whereas homozygous FH results in more severe LDL cholesterol elevation with death by 20 years of age. We present here the case of an African-American female FH patient presenting with a myocardial infarction at the age of 48, recurrent angina pectoris and numerous coronary artery stents. Her pretreated LDL cholesterol levels were more typical of a homozygous FH pattern and she was resistant to conventional lipid-lowering treatment, yet her other clinical parameters were not necessarily consistent with homozygous FH. Genetic testing revealed two LDLR variants on the same chromosome: one a novel missense mutation in exon 14 (Cys681Gly) and the other a promoter variant (IVS1-217C>T) previously shown to result in increased LDLR transcription. Disease-associated PCSK9 or APOB mutations were not identified in this individual. Overall, her genetic and clinical profile suggests that enhanced expression of the mutant LDLR allele resulted in a severe phenotype with characteristics of both heterozygous and homozygous FH.

PO5-135 ADVANCE IN MOLECULAR DIAGNOSIS OF FAMILIAL HYPERCHOLESTEROLEMIA IN THE CZECH POPULATION: IDENTIFICATION AND CHARACTERIZATION OF LARGE REARRANGEMENTS IN LDLR GENE

Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in the gene encoding the low density lipoprotein receptor (LDLR). Mutations in the LDLR gene were diagnosed using exon by exon screening methods based on individual exon amplification, DHPLC analysis and sequencing. Recently, a novel method for genomic quantification, Multiplex Ligation-dependent Probe Amplification (MLPA), has been developed. In order to explore the possibility that whole exon deletions or duplications could be the causative mutation on some yet uncharacterized mutant LDLR alleles, we used MLPA technique in this study.Six different large deletions and three duplications were found, 3 of which (EX9_15del-14kb, EX9_14del-9.5kb and EX2_6dup-8.5kb) were prevalent in FH individuals from the Czech population.

Retention of Mutant Low Density Lipoprotein Receptor in Endoplasmic Reticulum (ER) Leads to ER Stress

Familial hypercholesterolemia is an autosomal dominant disease caused by mutations in the gene encoding the low density lipoprotein receptor (LDLR). More than 50% of these mutations lead to receptor proteins that are completely or partly retained in the endoplasmic reticulum (ER). The mechanisms involved in the intracellular processing and retention of mutant LDLR are poorly understood. In the present study we show that the G544V mutant LDLR associates with the chaperones Grp78, Grp94, ERp72, and calnexin in the ER of transfected Chinese hamster ovary cells. Retention of the mutant LDLR was shown to cause ER stress and activation of the unfolded protein response. We observed a marked increase in the activity of two ER stress sensors, IRE1 and PERK. These results show that retention of mutant LDLR in ER induces cellular responses, which might be important for the clinical outcome of familial hypercholesterolemia.

Triglyceride-rich lipoprotein metabolism in unique VLDL receptor, LDL receptor, and LRP triple-deficient mice

The very low density lipoprotein receptor (VLDLR), low density lipoprotein receptor (LDLR), and low density lipoprotein receptor-related protein (LRP) are the three main apolipoprotein E-recognizing endocytic receptors involved in the clearance of triglyceride (TG)-rich lipoproteins from plasma. Whereas LDLR deficiency in mice results in the accumulation of plasma LDL-sized lipoproteins, VLDLR or LRP deficiency alone only minimally affects plasma lipoproteins. To investigate the combined effect of the absence of these receptors on TG-rich lipoprotein levels, we have generated unique VLDLR, LDLR, and LRP triple-deficient mice. Compared with wild-type mice, these mice markedly accumulated plasma lipids and lipases. These mice did not show aggravated hyperlipidemia compared with LDLR and LRP double-deficient mice, but plasma TG was increased after high-fat diet feeding. In addition, these mice showed a severely decreased postprandial TG clearance typical of VLDLR-deficient (VLDLR-/-) mice. Collectively, although VLDLR deficiency in LRP- and LDLR-/- mice does not aggravate hyperlipidemia, these triple-deficient mice represent a unique model of markedly delayed TG clearance on a hyperlipidemic background.

The molecular basis of familial hypercholesterolaemia in Turkish patients

Familial hypercholesterolaemia (FH) is an autosomal dominant disorder of lipoprotein metabolism. In the majority of patients FH is caused by mutations in the gene for the low-density lipoprotein receptor (LDLR), and to date more than 700 mutations have been reported worldwide. In this study, 36 paediatric patients with a clinical diagnosis of FH (20 homozygous and 16 heterozygotes) were screened for mutations in the LDLR gene. Each exon, with intron–exon junctions, was screened by capillary fluorescent SSCP (F-SSCP) and heteroduplex analysis. Samples showing different band patterns were sequenced. Ten novel (including three frame shift small deletions or insertions) and seven known mutations were detected. A total of 37 out of the predicted 56 FH-causing alleles were identified (66.1%). No patients with the R3500Q mutation in the APOB gene were found. W556R was the most common mutation, explaining 21.4% of the predicted defective LDLR alleles. The novel sequence changes were deemed to be pathogenic if they altered a conserved amino acid (L143P, D147E, Q233H-C234G, C347G) or occurred in or close to a splice site (IVS 16 + 5) and were absent in DNA from 50 healthy Turkish subjects. These data confirm the genetic heterogeneity of FH in Turkey, and demonstrate the usefulness of F-SSCP for mutation detection.

Rapid detection of GYPA, LDLR, HBGG, D7S8 and GC alleles by real-time fluorescence PCR

We developed the allele-specific TaqMan polymerase chain reaction (AS-TaqMan PCR) and SYBR Green PCR assays for detecting glycophorin A (GYPA), low-density lipoprotein receptor (LDLR), hemoglobin G (HBGG), D7S8 and group-specific component (GC) alleles. We improved the specificity of detecting a nucleotide substitution by introducing the additional mismatches at position 2 (3 in GYPA). The differences between threshold cycle (Ct) values of different genotypes on each of the loci were statistically significantly different. All the genotypes agreed with the results using the AmpliType PM+DQA1 PCR Amplification and Typing kit. The AS-TaqMan PCR and SYBR Green PCR assays are simple, rapid, and accurate, as well as suitable for high-throughput applications in a forensic investigation.

Impact of genetic defects on atherosclerosis in patients suspected of familial hypercholesterolaemia.

Among patients with severe hypercholesterolaemia and a family history of early cardiovascular disease, we assessed whether patients with mutations of low-density lipoprotein (LDL) receptor and apolipoprotein B genes related to familial hypercholesterolaemia (FH) have a different degree of atherosclerosis than those without such mutations. In our lipid clinics, 273 patients were selected on the basis of a severe hypercholesterolaemia (cholesterol above 95th percentile) and a family history of early cardiovascular disease. By molecular genetic test, 122 patients were classified as FH. Atherosclerosis was evaluated by the ultrasonographic measurement of intima-media thickness (IMT) in the carotid and femoral arteries. Despite the fact that non-FH individuals had a higher prevalence of obesity, hypertension, diabetes and hypertriglyceridaemia, FH individuals had significantly greater carotid and femoral IMT than non-FH patients: difference between carotid and femoral IMT, respectively, 0.19 mm (95% CI, 0.08-0.29; P < 0.001) and 0.20 mm (95% CI, 0.09-0.35; P = 0.001), respectively. These differences remained statistically significant after adjustment for the various risk factors as well as in sub-analysis restricted to the patients with LDL-cholesterol between 240 and 300 mg dL-1 (range with similar distribution in the two groups). When classified according to the severity of their mutations, FH individuals with null LDL receptor allele tended to have thicker carotid IMT than FH individuals carrying the LDL receptor-defective allele. Among patients with severe hypercholesterolaemia and a family history of early cardiovascular disease, the presence of a genetically ascertained FH is associated with a higher degree of atherosclerosis. This suggests that molecular genetic identification of FH may be helpful to evaluate better the coronary heart disease risk in these patients.

Co-segregation of elevated LDL with a novel mutation (D92K) of the LDL receptor in a kindred with multiple lipoprotein abnormalities.

Factors predisposing to the phenotypic features of familial combined hyperlipidemia have not been clearly defined. In the course of investigating familial coronary artery disease in Utah, we identified a three-generation family in which multiple members were affected with type IIa hyperlipoproteinemia (HLP IIa), type IIb hyperlipoproteinemia (HLP IIb), or type IV hyperlipoproteinemia (HLP IV). Because several family members had relatively severe low-density lipoprotein (LDL) cholesterol elevation, in order to dissect the possible contribution to the plasma lipoprotein abnormalities in this pedigree, we identified a novel point mutation in the low-density lipoprotein receptor (LDLR) gene, a G-to-A transition at nucleotide position 337 in exon 4. This change substituted lysine for glutamic acid at codon 92 (D92K) of the LDL receptor. By means of mutant allele-specific amplification we determined that the mutation co-segregated with elevated cholesterol and LDL cholesterol in the plasma of family members with HLP IIa and HLP IIb, but not with the elevated plasma triglycerides seen in HLP IIb and HLP IV patients. Thus, in families with apparent familial combined hyperlipidemia, a defective LDLR allele and other genetic or environmental factors that elevate plasma triglycerides may account for the multiple lipid phenotypes observed in this kindred.

Expression of an LDL receptor allele with two different mutations (E256K and I402T)

Aims—To investigate the disease causing event in patients with familial hypercholesterolaemia, carrying two mutations each, E256K in exon 6 and I402T in exon 9, of the gene encoding the low...

Overexpression and Accumulation of Apolipoprotein E as a Cause of Hypertriglyceridemia

The molecular mechanisms of hypertriglyceridemia (HTG), a common lipid metabolic disorder in humans, often of genetic origin, are not well understood. In studying the effect of apolipoprotein (apo) E on the metabolism of triglyceride-rich lipoproteins, we found that expressing high plasma levels of human apoE3 in transgenic mice lacking endogenous mouse apoE caused HTG. These transgenic animals had 3-fold higher plasma triglyceride levels, higher very low density lipoproteins (VLDL), and lower high density lipoproteins than did nontransgenics. Removing one or both low density lipoprotein receptor alleles in the apoE3-overexpressing mice caused severe HTG (8-11-fold over nontransgenics) and increased VLDL and decreased low and high density lipoproteins, and apoE3-enriched VLDL were markedly depleted in apoC-II. At least two mechanisms could explain HTG associated with apoE3 overexpression: stimulated VLDL triglyceride production and impaired VLDL lipolysis. The apoE3 mice with HTG had a 50% increase in hepatic VLDL triglyceride production. Furthermore, overexpression of apoE (E2, E3, or E4) in cultured hepatocytes (McA-RH7777 cells) correlated positively with secretion of VLDL into the medium. However, apoE3 overexpression-associated HTG was only partially explained by VLDL overproduction, as lipoprotein lipase-mediated VLDL lipolysis was also decreased 20-86% depending on apoE3 levels, most likely by displacing or masking apoC-II on the particles. In human subjects, HTG correlated positively with increased VLDL triglyceride and plasma and VLDL apoE levels. However, plasma and VLDL apoE correlated negatively with VLDL apoC-II levels and lipoprotein lipase-mediated VLDL lipolysis. Thus, optimal expression of apoE is crucial for normal metabolism of triglyceride-rich lipoproteins, and overexpression and/or accumulation of apoE may contribute to HTG by stimulating VLDL triglyceride production and by impairing VLDL lipolysis. The apoE3-overexpressing mice will be useful for studying the pathophysiology of this disorder.