Molecular characterization of Polish patients with familial hypercholesterolemia: novel and recurrentLDLR mutations

Autosomal dominant hypercholesterolemia (ADH) is caused by mutations in the genes coding for the low-density lipoprotein receptor (LDLR), apolipoprotein B-100 (APOB), or proprotein convertase subtilisin/kexin type 9 (PCSK9). In this study, a molecular analysis ofLDLR andAPOB was performed in a group of 378 unrelated ADH patients, to explore the mutation spectrum that causes hypercholesterolemia in Poland. All patients were clinically diagnosed with ADH according to a uniform protocol and internationally accepted WHO criteria. Mutational analysis included all exons, exon-intron boundaries and the promoter sequence of theLDLR, and a fragment of exon 26 ofAPOB. Additionally, the MLPA technique was applied to detect rearrangements withinLDLR. In total, 100 sequence variations were identified in 234 (62%) patients. WithinLDLR, 40 novel and 59 previously described sequence variations were detected. Of the 99LDLR sequence variations, 71 may be pathogenic mutations. The most frequentLDLR alteration was a point mutation p.G592E detected in 38 (10%) patients, followed by duplication of exons 4–8 found in 16 individuals (4.2%). Twenty-five cases (6.6%) demonstrated the p.R3527Q mutation ofAPOB. Our findings imply that major rearrangements of theLDLR gene as well as 2 point mutations (p.G592E inLDLR and p.R3527Q inAPOB) are frequent causes of ADH in Poland. However, the heterogeneity ofLDLR mutations detected in the studied group confirms the requirement for complex molecular studies of Polish ADH patients.     

Use of Targeted Exome Sequencing in Genetic Diagnosis of Chinese Familial Hypercholesterolemia

Familial hypercholesterolemia is an autosomal dominant inherited disease characterized by elevated plasma low-density lipoprotein cholesterol (LDL-C). It is mainly caused by mutations of the low-density lipoprotein receptor (LDLR) gene. Currently, the methods of whole genome sequencing or whole exome sequencing for screening mutations in familial hypercholesterolemia are not applicable in China due to high cost. We performed targeted exome sequencing of 167 genes implicated in the homozygous phenotype of a proband pedigree to identify candidate mutations, validated them in the family of the proband, studied the functions of the mutant protein, and followed up serum lipid levels after treatment. We discovered that exon 9 c.1268 T>C and exon 8 c.1129 T>G compound heterozygous mutations in the LDLR gene in the proband derived from the mother and father, respectively, in which the mutation of c.1129 T>G has not been reported previously. The mutant LDL-R protein had 57% and 52% binding and internalization functions, respectively, compared with that of the wild type. After 6 months of therapy, the LDL-C level of the proband decreased by more than 50% and the LDL-C of the other family members with heterozygous mutation also reduced to normal. Targeted exome sequencing is an effective method for screening mutation genes in familial hypercholesterolemia. The exon 8 and 9 mutations of the LDLR gene were pedigree mutations. The functions of the mutant LDL-R protein were decreased significantly compared with that of the wild type. Simvastatin plus ezetimibe was proven safe and effective in this preschool-age child.     

Transcriptomic Identification of ADH1B as a Novel Candidate Gene for Obesity and Insulin Resistance in Human Adipose Tissue in Mexican Americans from the Veterans Administration Genetic Epidemiology Study (VAGES)

Type 2 diabetes (T2D) is a complex metabolic disease that is more prevalent in ethnic groups such as Mexican Americans, and is strongly associated with the risk factors obesity and insulin resistance. The goal of this study was to perform whole genome gene expression profiling in adipose tissue to detect common patterns of gene regulation associated with obesity and insulin resistance. We used phenotypic and genotypic data from 308 Mexican American participants from the Veterans Administration Genetic Epidemiology Study (VAGES). Basal fasting RNA was extracted from adipose tissue biopsies from a subset of 75 unrelated individuals, and gene expression data generated on the Illumina BeadArray platform. The number of gene probes with significant expression above baseline was approximately 31,000. We performed multiple regression analysis of all probes with 15 metabolic traits. Adipose tissue had 3,012 genes significantly associated with the traits of interest (false discovery rate, FDR ≤ 0.05). The significance of gene expression changes was used to select 52 genes with significant (FDR ≤ 10^-4) gene expression changes across multiple traits. Gene sets/Pathways analysis identified one gene, alcohol dehydrogenase 1B (ADH1B) that was significantly enriched (P < 10^-60) as a prime candidate for involvement in multiple relevant metabolic pathways. Illumina BeadChip derived ADH1B expression data was consistent with quantitative real time PCR data. We observed significant inverse correlations with waist circumference (2.8 x 10^-9), BMI (5.4 x 10^-6), and fasting plasma insulin (P < 0.001). These findings are consistent with a central role for ADH1B in obesity and insulin resistance and provide evidence for a novel genetic regulatory mechanism for human metabolic diseases related to these traits.     

LRP4 induces extracellular matrix productions and facilitates chondrocyte differentiation

Endochondral ossification is an essential step for skeletal development, which requires chondrocyte differentiation in growth cartilage. The low-density lipoprotein receptor-related protein 4 (LRP4), a member of LDLR family, is an inhibitor for Wnt signaling, but its roles in chondrocyte differentiation remain to be investigated. Here we found by laser capture microdissection that LRP4 expression was induced during chondrocyte differentiation in growth plate. In order to address the roles, we overexpressed recombinant human LRP4 or knocked down endogenous LRP4 by lentivirus in mouse ATDC5 chondrocyte cells. We found that LRP4 induced gene expressions of extracellular matrix proteins of type II collagen (Col2a1), aggrecan (Acan), and type X collagen (Col10a1), as well as production of total proteoglycans in ATDC5 cells, whereas LRP4 knockdown had opposite effects. Interestingly, LRP4-knockdown reduced mRNA expression of Sox9, a master regulator for chondrogenesis, as well as Dkk1, an extracellular Wnt inhibitor. Analysis of Wnt signaling revealed that LRP4 blocked the Wnt/β-catenin signaling activity in ATDC5 cells. Finally, the reduction of these extracellular matrix productions by LRP4-knockdown was rescued by a β-catenin/TCF inhibitor, suggesting that LRP4 is an important regulator for extracellular matrix productions and chondrocyte differentiation by suppressing Wnt/β-catenin signaling.     

LDL-R AvaII and NcoI Polymorphisms: An Indirect Risk Factor for Coronary Heart Disease Among a Mendelian Population of Delhi, India

AvaII and NcoI polymorphisms in the low-density lipoprotein receptor (LDL-R) gene are reported to alter cholesterol levels. Although found to be highly polymorphic worldwide, these mutations have not been validated in any Indian population. This case–control association study was conducted in an endogamous business community of Delhi. Blood samples from 100 cases and 100 age- and sex-matched controls belonging to the same ethnic group were subjected to biochemical and molecular analyses. Medical history and anthropometric measurements were taken from all the enrolled subjects. Linkage disequilibrium between the two polymorphisms was found to be significant (P = 0.0016). Significant variability was observed for the AvaII polymorphism among cases concerning waist–hip ratio, serum triglyceride, and low-density lipoprotein, which in turn was found to be associated with coronary heart disease.     

A novel loop-mediated isothermal amplification-based genotyping method and its application for identifying proprotein convertase subtilisin/kexin type 9 variants in familial hypercholesterolemia

BackgroundProprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating low-density lipoprotein levels in plasma. While PCSK9 variants are causatively associated with familial hypercholesterolemia (FH), additional genotyping methods for FH targeting PCSK9 variants are required in a clinical setting. Loop-mediated isothermal amplification (LAMP) is a unique amplification method that amplifies a target gene under isothermal conditions (60–65 °C). However, a robust standardized method has not yet been established for LAMP-based genetic screening tests for genetic diseases, including FH. The present study aimed to develop a novel modification of the LAMP method designed to genotype single nucleotide variants (SNVs) and to apply it for the detection of PCSK9 variants.MethodsUsing short quenching probes (≤ 10 nucleotides) for the loop structures of LAMP amplicons, accurate detection of SNVs was verified separately for each allele, without any additional procedures, within 3 h. The diagnostic performance of this method in detecting PCSK9 variants was validated in FH patients.ResultsAll PCSK9 variants tested via conventional sequencing in FH patients were successfully detected using this novel LAMP method.ConclusionsWe developed a LAMP-based genotyping method to detect PCSK9 variants in FH. Compared to conventional sequencing, our genotyping method is relatively convenient and time-efficient and is suitable for the screening of FH in clinical settings. Future studies on various genes are also warranted.     

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.     

Cloning of a novel member of the low-density lipoprotein receptor family

A gene encoding a novel transmembrane protein was identified by DNA sequence analysis within the insulin-dependent diabetes mellitus (IDDM) locus IDDM4 on chromosome 11q13. Based on its chromosomal position, this gene is a candidate for conferring susceptibility to diabetes. The gene, termed low-density lipoprotein receptor related protein 5 (LRP5), encodes a protein of 1615 amino acids that contains conserved modules which are characteristic of the low-density lipoprotein (LDL) receptor family. These modules include a putative signal peptide for protein export, four epidermal growth factor (EGF) repeats with associated spacer domains, three LDL-receptor (LDLR) repeats, a single transmembrane spanning domain, and a cytoplasmic domain. The encoded protein has a unique organization of EGF and LDLR repeats; therefore, LRP5 likely represents a new category of the LDLR family. Both human and mouse LRP5 cDNAs have been isolated and the encoded mature proteins are 95% identical, indicating a high degree of evolutionary conservation.     

Causal Role of Alcohol Consumption in an Improved Lipid Profile: The Atherosclerosis Risk in Communities (ARIC) Study

Introduction

Health benefits of low-to-moderate alcohol consumption may operate through an improved lipid profile. A Mendelian randomization (MR) approach was used to examine whether alcohol consumption causally affects lipid levels.

Methods

This analysis involved 10,893 European Americans (EA) from the Atherosclerosis Risk in Communities (ARIC) study. Common and rare variants in alcohol dehydrogenase and acetaldehyde dehydrogenase genes were evaluated for MR assumptions. Five variants, residing in the ADH1B, ADH1C, and ADH4 genes, were selected as genetic instruments and were combined into an unweighted genetic score. Triglycerides (TG), total cholesterol, high-density lipoprotein cholesterol (HDL-c) and its subfractions (HDL2-c and HDL3-c), low-density lipoprotein cholesterol (LDL-c), small dense LDL-c (sdLDL-c), apolipoprotein B (apoB), and lipoprotein (a) (Lp(a)) levels were analyzed.

Results

Alcohol consumption significantly increased HDL2-c and reduced TG, total cholesterol, LDL-c, sdLDL-c, and apoB levels. For each of these lipids a non-linear trend was observed. Compared to the first quartile of alcohol consumption, the third quartile had a 12.3% lower level of TG (p < 0.001), a 7.71 mg/dL lower level of total cholesterol (p = 0.007), a 10.3% higher level of HDL2-c (p = 0.007), a 6.87 mg/dL lower level of LDL-c (p = 0.012), a 7.4% lower level of sdLDL-c (p = 0.037), and a 3.5% lower level of apoB (p = 0.058, p_overall = 0.022).

Conclusions

This study supports the causal role of regular low-to-moderate alcohol consumption in increasing HDL2-c, reducing TG, total cholesterol, and LDL-c, and provides evidence for the novel finding that low-to-moderate consumption of alcohol reduces apoB and sdLDL-c levels among EA. However, given the nonlinearity of the effect of alcohol consumption, even within the range of low-to-moderate drinking, increased consumption does not always result in a larger benefit.     

Allelic variation at alcohol metabolism genes (<Emphasis Type="Italic">ADH1B</Emphasis>,<Emphasis Type="Italic"> ADH1C</Emphasis>,<Emphasis Type="Italic"> ALDH2</Emphasis>) and alcohol dependence in an American Indian population

Enzymes encoded by two gene families, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), mediate alcohol metabolism in humans. Allelic variants have been identified that alter metabolic rates and influence risk for alcoholism. Specifically, ADH1B*47His (previously ADH2-2) and ALDH2-2 have been shown to confer protection against alcoholism, presumably through accumulation of acetaldehyde in the blood and a resultant 'flushing response' to alcohol consumption. In the current study, variants at ADH1B (previously ADH2), ADH1C (previously ADH3), and ALDH2 were assayed in DNA extracts from participants belonging to a Southwest American Indian tribe (n=490) with a high prevalence of alcoholism. Each subject underwent a clinical interview for diagnosis of alcohol dependence, as well as evaluation of intermediate phenotypes such as binge drinking and flushing response to alcohol consumption. Detailed haplotypes were constructed and tested against alcohol dependence and related intermediate phenotypes using both association and linkage analysis. ADH and ALDH variants were also assayed in three Asian and one African population (no clinical data) in order to provide an evolutionary context for the haplotype data. Both linkage and association analysis identified several ADH1C alleles and a neighboring microsatellite marker that affected risk of alcohol dependence and were also related to binge drinking. These data strengthen the support for ADH as a candidate locus for alcohol dependence and suggest further productive study.     

Disease-Corrected Hepatocyte-Like Cells from Familial Hypercholesterolemia-Induced Pluripotent Stem Cells

The generation of human induced pluripotent stem cells (hiPSCs) from an individual patient provides a unique tool for disease modeling, drug discovery, and cell replacement therapies. Patient-specific pluripotent stem cells can be expanded in vitro and are thus suitable for genetic manipulations. To date, several genetic liver disorders have been modeled using patient-specific hiPSCs. Here, we present the generation of corrected hepatocyte-like cells (HLCs) from hiPSCs of a familial hypercholesterolemia (FH) patient with a homozygous mutation in the low-density lipoprotein receptor (LDLR) gene. We generated hiPSCs from a patient with FH with the mutated gene encoding a truncated non-functional receptor. In order to deliver normal LDLR to the defective cells, we used a plasmid vector carrying the normal receptor ORF to genetically transform the hiPSCs. The transformed cells were expanded and directed toward HLCs. Undifferentiated defective hiPSCs and HLCs differentiated from the defective hiPSCs did not have the ability to uptake labeled low-density lipoprotein (LDL) particles. The differentiated transformed hiPSCs showed LDL-uptake ability and the correction of disease phenotype as well as expressions of hepatocyte-specific markers. The functionality of differentiated cells was also confirmed by indo-cyanine green (ICG) uptake assay, PAS staining, inducible cyp450 activity, and oil red staining. These data suggest that hiPSC technology can be used for generation of disease-corrected, patient-specific HLCs with potential value for disease modeling and drug discovery as well as cell therapy applications in future.     

Conditioning Factors for High Cardiovascular Risk in Patients with Cushing Syndrome

Objective: To characterize the alterations in carbohydrate and lipoprotein metabolism, to evaluate markers of lipoprotein functionality, and to identify the presence of novel atherogenic risk factors in patients with Cushing syndrome (CS) in comparison with sex- and age-matched controls.Methods: In an open, cross-sectional study, 32 nontreated patients with active CS were consecutively recruited from the Endocrinology Service at “José de San Martín” Clinical Hospital, University of Buenos Aires, Argentina, between April 11, 2010 and December 11, 2012. The patients were compared with sex- and age-matched controls.Results: Versus controls, patients with CS presented with excess weight, central obesity, and hypercortisolism. They also exhibited an insulin-resistant state, with high resistin levels (median [interquartile range], 16 [10 to 22] ng/mL versus 6 [5 to 9] ng/mL; P<.0001), a more atherogenic lipoprotein profile, high oxidized low-density lipoprotein levels (oxLDL; mean ± SD, 100 ± 31 U/L versus 75 ± 32 U/L; P<.05) and high sensitive C-reactive protein levels (median [interquartile range], 1.2 [0.6 to 3.1] mg/L versus 0.6 [0.3 to 1.1] mg/L; P<.05), and increased leukocyte count (mean ± SD, 9.5 ± 2.6 × 10³ cells/μL versus 6.5 ± 1.4 × 10³ cells/μL; P<.0001). Multivariate analyses showed that the increase in waist circumference was associated with both the diagnosis of CS and the degree of insulin resistance. Resistin concentration was related to a greater extent to the diagnosis of CS than to homeostasis model assessment–insulin resistance. Triglyceride and oxLDL levels were only significantly associated with the diagnosis of CS.Conclusion: Hypercortisolism is related to the increase observed in triglycerides and oxLDL levels, and, in combination with insulin resistance, acts to increase waist circumference and amplify the inflammatory process, key factors for the development of cardiovascular disease.Abbreviations: apo = apolipoprotein ARE = arylesterase CETP = cholesteryl ester transfer protein CRP = C-reactive protein CS = Cushing syndrome CV = coefficient of variation HDL = high-density lipoprotein HDL-C = high-density-lipoprotein cholesterol HOMA = homeostasis model assessment LDL = low-density lipoprotein LDL-C = low-density-lipoprotein cholesterol Lp-PLA₂ = lipoprotein-associated phospholipase A₂ oxLDL = oxidized LDL PON = paraoxonase TG = triglyceride     

The T705I mutation of the low density lipoprotein receptor gene (FH Paris-9) does not cause familial hypercholesterolemia

Familial hypercholesterolemia (FH) is a genetic disease caused by mutations in the low-density lipoprotein receptor gene. Among the more than 200 mutations so far identified, the T705I substitution in exon 15, designated FH-Paris 9, has been previously described as an FH-causing mutation. During the course of denaturing gradient gel electrophoretic screening of exon 15 we have identified the T705I single-base substitution not only in an FH family but also in a control, normocholesterolemic population. Therefore, we conclude that FH-Paris 9 is a missense mutation not associated with FH. Received: 5 March 1996 / Revised: 28 July 1996     

Common mutations of familial hypercholesterolemia patients in Taiwan: characteristics and implications of migrations from southeast China

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in low-density lipoprotein receptor (LDLR), apolipoprotein B-100 (APOB), and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. This study investigated FH patients carrying common mutations in Taiwan and compared them to FH southeastern Asians. Causal FH mutations were identified by exon-by-exon sequencing with/without multiplex ligation-dependent probe amplification among 208 Taiwanese with clinically diagnosed FH. Haplotype analyses among probands and family members were undertaken using TaqMan® Assays. Totally, LDLR mutations were found in 118 probands, consisting of 61 different loci, and APOB 10579C>T mutations in 12 probands. Three mutations (64delG, 1661C>T, and 2099A>G) were novel. LDLR 986G>A (13.1%), 1747C>T (10.8%), and APOB 10579C>T (9.2%) were common mutations with no differences in phenotypes. LDLR 1747C>T associated with one haplotype (CAAGCCCCATGG/(dTA)n-112nt); LDLR 986G>A with two. APOB 10579C>T associated with the same LDLR binding-domain pattern in Taiwanese and southeastern Asians. We concluded that LDLR 986G>A, 1747C>T and APOB 10579C>T are common mutations, with combined frequency of approximately 33%. The presence of different haplotypes associated with FH common mutations in Taiwan indicates multiple historical migrations, probable multiple recurrent origins from southern China, and haplotype homologies reflect the presence of common ancestors in southern China.     

Genetic Variants Associated with Gestational Hypertriglyceridemia and Pancreatitis

Severe hypertriglyceridemia is a well-known cause of pancreatitis. Usually, there is a moderate increase in plasma triglyceride level during pregnancy. Additionally, certain pre-existing genetic traits may render a pregnant woman susceptible to development of severe hypertriglyceridemia and pancreatitis, especially in the third trimester. To elucidate the underlying mechanism of gestational hypertriglyceridemic pancreatitis, we undertook DNA mutation analysis of the lipoprotein lipase (LPL), apolipoprotein C2 (APOC2), apolipoprotein A5 (APOA5), lipase maturation factor 1 (LMF1), and glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) genes in five unrelated pregnant Chinese women with severe hypertriglyceridemia and pancreatitis. DNA sequencing showed that three out of five patients had the same homozygous variation, p.G185C, in APOA5 gene. One patient had a compound heterozygous mutation, p.A98T and p.L279V, in LPL gene. Another patient had a compound heterozygous mutation, p.A98T & p.C14F in LPL and GPIHBP1 gene, respectively. No mutations were seen in APOC2 or LMF1 genes. All patients were diagnosed with partial LPL deficiency in non-pregnant state. As revealed in our study, genetic variants appear to play an important role in the development of severe gestational hypertriglyceridemia, and, p.G185C mutation in APOA5 gene appears to be the most common variant implicated in the Chinese population. Antenatal screening for mutations in susceptible women, combined with subsequent interventions may be invaluable in the prevention of potentially life threatening gestational hypertriglyceridemia-induced pancreatitis.     

Benzophenones and xanthones from Garcinia cantleyana var. cantleyana and their inhibitory activities on human low-density lipoprotein oxidation and platelet aggregation

Three benzophenones, 2,6,3′,5′-tetrahydroxybenzophenone (1), 3,4,5,3′,5′-pentahydroxybenzophenone (3) and 3,5,3′,5′-tetrahydroxy-4-methoxybenzophenone (4), as well as a xanthone, 1,3,6-trihydroxy-5-methoxy-7-(3′-methyl-2′-oxo-but-3′-enyl)xanthone (9), were isolated from the twigs of Garcinia cantleyana var. cantleyana. Eight known compounds, 3,4,5,3′-tetrahydroxy benzophenone (2), 1,3,5-trihydroxyxanthone (5), 1,3,8-trihydroxyxanthone (6), 2,4,7-trihydroxyxanthone (7), 1,3,5,7-tetrahydroxyxanthone (8), quercetin, glutin-5-en-3β-ol and friedelin were also isolated. The structures of the compounds were elucidated by spectroscopic methods. The compounds were investigated for their ability to inhibit low-density lipoprotein (LDL) oxidation and platelet aggregation in human whole blood in vitro. Most of the compounds showed strong antioxidant activity with compound 8 showing the highest inhibition with an IC₅₀ value of 0.5 μM, comparable to that of probucol. Among the compounds tested, only compound 4 exhibited strong inhibitory activity against platelet aggregation induced by arachidonic acid (AA), adenosine diphosphate (ADP) and collagen. Compounds 3, 5 and 8 showed selective inhibitory activity on platelet aggregation induced by ADP.     

An enhancer-blocking element regulates the cell-specific expression of alcohol dehydrogenase 7

The class IV alcohol dehydrogenase gene ADH7 encodes an enzyme that is involved in ethanol and retinol metabolism. ADH7 is expressed mainly in the upper gastrointestinal tract and not in the liver, the major site of expression of the other closely related ADHs. We identified an intergenic sequence (iA1C), located between ADH7 and ADH1C, that has enhancer-blocking activity in liver-derived HepG2 cells that do not express their endogenous ADH7. This enhancer blocking function was cell- and position-dependent, with no activity seen in CP-A esophageal cells that express ADH7 endogenously. iA1C function was not specific to the ADH enhancers; it had a similar cell-specific effect on the SV40 enhancer. The CCCTC-binding factor (CTCF), an insulator binding protein, bound iA1C in HepG2 cells but not in CP-A cells. Our results suggest that in liver-derived cells, iA1C blocks the effects of ADH enhancers and thereby contributes to the cell specificity of ADH7 expression.