Familial Hypercholesterolemia Genetic and Functional Studies in Taiwanese Population
Date Issued
2005
Date
2005
Author(s)
DOI
zh-TW
Abstract
【Background】
Genetic Characterizations of ADH patients
Autosomal dominant hypercholesterolemia (ADH) is an inherited disorder of cholesterol metabolism characterized by a high concentration of plasma LDL-C, deposition of cholesterol in tendons and skin, and increased risk of premature coronary heart disease (CHD). ADH is most commonly caused by mutations in the LDL receptor (LDLR) gene, which can lead to reduced hepatic clearance of LDL from the blood. The estimated prevalence of LDLR gene mutation is 1 per 500 in its heterozygous form. To date, more than 800 mutations for the LDLR gene have been reported. ADH can also be caused by certain mutations in apolipoprotein B (APOB) gene, which encodes the ligand for LDLR, named familial defective apolipoprotein B (FDB). FDB occurs with a prevalence of 1 per 1000 in most populations. Until recently, a third locus responsible for ADH (FH3) was identified at 1p34.1-p32 in several large ADH kindreds without mutations in LDLR or APOB genes. The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene, localized to the third FH locus, has been proposed to be the third gene with pathogenic mutations accounting for ADH. PCSK9 encodes neural-apoptosis -regulated convertase (NARC-1), a novel protein that may play a crucial role in cholesterol homeostasis, though the exact molecular mechanisms are still obscure.
Although heterozygous ADH is presumed to be a common disorder resulting in atherosclerosis in Asians, there is very limited epidemiologic and genetic data with regard to Taiwanese ADH patients. To establish the molecular basis of ADH in Taiwan, we investigated the genes of LDLR, APOB and PCSK9 for mutations in Taiwanese ADH patients.
Hypercholesterolemia has been recognized as a major risk factor for the development of atherosclerosis and coronary heart disease. There have been abundant evidences suggesting hypercholesterolemia may impair endothelial function, accelerate the progression of atherosclerosis, and ultimately increase the risk of ischemia in myocardium and many other end organs. In this study, we would like to evaluate the effect of hypercholesterolemia on cardiovascular system, with particular interest on endothelial function, intima-media thickness and myocardial function, in patients with ADH
【Aims, Materials and Methods】
Patients
Patients attending the Lipid Clinic at National Taiwan University Hospital (NTUH), diagnosed as ADH were recruited in our study. The diagnostic criteria of ADH included (1)fasting plasma total cholesterol and LDL-C levels above 95th percentiles for adult Taiwanese after adjust with age and gender and triglycerides<220 mg/dl (2.5 mmol/l), and (2) presence of tendon xanthomata/xantholesma/corneal arcus or premature CHD in index case or first degree relative, or a family history of hypercholesterolemia consistent with an autosomal dominant inheritance. Patients with secondary causes for hypercholesterolemia, such as hypothyroidism, renal or hepatic disease, were excluded.
Lipid measurements
Blood samples from fasting patients without concurrent lipid-lowering therapy were obtained for measurements. The concentration of plasma total cholesterol, high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) were determined with commertially available kits (Boehringer Mannheim). LDL-C was estimated with the aid of the Friedewald formula .
DNA Preparation
Genomic DNA was isolated from EDTA whole blood with the Puregene DNA Isolation Kit (Gentra Systems, Inc., Minneapolis, USA) according to the manufacturer’s instructions.
PCR
PCR amplification of the LDLR gene (including the promoter, 18 coding exons and flanking intron regions), APOB gene (the exon 26 of APOB gene containing condons 3473-3561), and the PCSK9 gene (the 12 exons and flanking intron regions) were performed with primers provided in Table 1. Each PCR mixture, with total volume of 25 mL, contained 50 ng of genomic DNA, 0.12 mM of each primer, 100 mM dNTPs, 0.5 units of AmpliTaq GoldTM enzyme (PE Applied Biosystems, Foster City, USA), and 2.5 mL of GeneAmp 10X buffer II (10 mM Tris-HCl, pH = 8.3, 50 mM KCl), in 2 mM MgCl2 as provided by the manufacturer. Amplification was performed in a multiblock system (MBS) thermocycler (ThermoHybaid, Ashford, UK). PCR amplification was performed with an initial denaturation step at 95℃ for 10 min, followed by 35 cycles consisting of denaturation at 94℃ for 30 sec, annealing at 55-57℃ for 60 sec (specific annealing temperature for each PCR product are listed in Table 1), extension at 72℃ for 30 sec, and then a final extension step at 72℃ for 10 min.
DHPLC Analysis
Mutation analysis was performed on a Transgenomic Wave Nucleic Acid Fragment Analysis System (Transgenomic Inc., San Jose, USA). Denaturing high performance liquid chromatography (DHPLC) was carried out on automated HPLC instrumentation equipped with a DNASep column (Transgenomic Inc.). DHPLC-grade acetonitrile (9017-03, JT Baker, Phillipsburg, NJ) and triethylammonium acetate (TEAA, Transgenomic Inc.,Crewe, UK) were used to constitute the mobile phase. The mobile phases comprised 0.05% acetonitrile in 0.1 M TEAA (eluent A) and 25% acetonitrile in 0.1 M TEAA (eluent B). For heteroduplex detection of crude PCR products, subjected to an additional 3-min 95℃ denaturing step followed by gradual reannealing from 95℃ to 65℃ over a period of 30 min prior to analysis, were eluted at a flow rate of 0.9 mL/min. The start- and end-points of the gradient obtained by mixing eluents A and B and the temperature required for successful resolution of heteroduplex molecules, were deduced from the WAVEmaker system control software version 4.1.42 (Transgenomic Inc.). Eight microliters of PCR product was injected for analysis in each running. The DHPLC temperatures for each PCR product are listed in Table 1. Heterozygous profiles were identified by visual inspection of the chromatograms on the basis of the appearance of additional earlier eluting peaks. Corresponding homozygous profiles show as only one peak.
Direct Sequencing Analysis
PCR products from index cases of ADH who showed abnormal DHPLC heteroduplex pattern compared with controls were sequenced. Amplicons were purified by solid-phase extraction and bidirectionally sequenced with the PE Biosystems Taq DyeDeoxy terminator cycle sequencing kit (PE Biosystems) according to the manufacturer’s instructions. Sequencing reactions were separated on a PE Biosystems 373A/3100 sequencer.
Carotid Artery Intima-Media Thickness (IMT)
With the use of a high-resolution B-mode ultrasonography (7.5MHz real-time B-mode scanner with HP SONO 1000 ultrasound system), we obtained 2 measurement of IMT on the far wall of both R’t and L’t CCA along a 1 cm section proximal to the carotid bulb. Carotid IMT was defined as the distance from the leading edge of the first echogenic (bright line) to the the leading edge of the second echogeinc line. Two measurements were done on each side of the CCA among 60 ADH patients and another 60 age-gender matched normocholesterolemic conrols.
FMD (flow-mediated dilatation) of Brachial Artery
Using a high-resolution B-mode ultrasonography (7.5MHz real-time B-mode
scanner with HP SONO 1000 ultrasound system), we obtained two measurement of brachial a. diameter in right antecubital fossa at baseline. Then we inflated blood pressure cuff up to 200mmHg or 50mmHg above baseline systolic BP to compress brachial artery for 5 minutes. After then, we release the cuff and measure brachial a. diameter again. FMD was defined as the percentage change of the post-compression brachial artery diameter from baseline.
Echocardiography Evaluation of the Myocardial Function
All studies were performed with a Hewlett-Packard Sonos 1000 system, with a 3.5 mHz duplex probe. Standard 2-dimensional and color flow Doppler images were obtained in all patients in the parasternal short-axis and apical views. Each patient underwent LV myocardial function assessment by conventional Doppler, tissue Doppler imaging (TDI), and calculated myocardial performance index (Tei index).
In conclusion, the genetic background of Taiwanese ADH patients is highly heterogeneous, consisting of a variety of different mutations in LDLR and APOB genes. However, there may exist some common mutations responsible for a significant portion of ADH population in Taiwan. The mutations of the PCSK9 gene seem not to play a significant role to cause ADH in Taiwanese. These observations reflect the heterogeneous ethnic origins of Taiwanese and a characterized mutation pattern that is different from those in other countries. A larger screening program is required to clarify the epidemiological features of ADH in Taiwan. In vitro expression study is also needed to confirm the functional implication of the newly identified mutations in ADH patients. We also demonstrated that hypercholesterolemia in FH subjects can lead to an increased carotid artery IMT and a higher risk of myocardial diastolic dysfunction.
Subjects
基因研究
心血管功能
Familial hypercholesterolemia
genetic study
Taiwan
Cardiovascular function
Type
text
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