台灣地區華人家族性高膽固醇血症之基因及心血管功能研究

Abstract

背景 高膽固醇血症會使冠狀動脈疾病、動脈硬化及中風的相對危險性大幅增加，而異型接合子家族性高膽固醇血症(heterozygous familial hypercholesterolemia，FH)是導致高膽固醇血症其中一個常見而重要的原因。家族性高膽固醇血症主要肇因於低密度脂蛋白受體(LDL receptor)或其配體(LDL receptor ligand) Apo-B 100，以及一新近發現之PCSK9基因變異，使低密度脂蛋白膽固醇(LDL cholesterol)代謝異常，臨床上常以高膽固醇血症、黃瘤(xanthoma)及早發性心肌梗塞來表現。家族性高膽固醇血症是屬於體染色體顯性(autosomal dominant)的遺傳性疾病，同型接合子家族性高膽固醇血症(homozygous FH)相當罕見，發生率約為百萬分之一，多半於年幼時即因併發心血管疾病而死亡；而異型接合子家族性高膽固醇血症則較為常見，據估計約每五百人中就有一人帶有相關的基因突變，其中約有一半在50歲之前就可能發生心血管疾病。因此，早期診斷家族性高膽固醇血症，並給予適當飲食及藥物治療，可以降低該族群日後發生心血管病變的機會，對於疾病預防及減少社會醫療成本上有相當大的幫助。目前關於台灣人家族性高膽固醇血症基因突變的研究仍然十分缺乏，本研究的目的在找出台灣地區華人家族性高膽固醇血症基因變異型式，並評估其心血管功能受到的影響。 目標，材料及方法 第一部份是關於家族性高膽固醇血症患者的基因變異研究，我們從台大醫院高血脂門診中篩選出符合家族性高膽固醇血症的患者作為指引個案，萃取指引個案及其一等親以內家屬之血液中淋巴球之DNA進行分析。先對包含低密度脂蛋白受體基因(LDLR)，Apo-B 100及PCSK9基因在內共33 DNA片段進行聚合酶鏈反應（polymerase chain reaction, PCR），以變性高效液相層析技術（DHPLC, denaturing high performance liquid chromatography）分析PCR產物中可能發生的相關突變或單核苷酸基因多形性（SNP, single nucleotide polymorphism），並對可能發生突變的DNA片段進行序列分析，來確認其基因變異的型式。 第二部份是評估家族性高膽固醇血症對人體心血管功能的影響。我們為所有收案之FH患者於臨床診斷時執行心血管功能檢查，包含頸動脈之內膜中膜層厚度(IMT, intima-media thickness)，肱動脈之內皮依賴性血管舒張功能(FMD, flow-mediated dilation)，心臟超音波之Tei指標，都普勒組織成像 (DTI, Doppler tissue imaging)及左心室舒張功能評估(LV diastolic function)，並與非高膽固醇血症之對照組個案比較，以評估家族性高膽固醇血症對其心血管功能之影響。 結果及結論 我們一共完成18個高膽固醇血症家族的基因及心血管功能分析，其中有11個家族(61.1%)確定帶有LDLR，APOB或PCSK9 gene的突變，包含了LDLR的6種突變(其中delAT1954及G>C1586+5兩者為novel mutation)及APOB的兩個點突變(其中T3540M為novel mutation)。LDLR的基因變異方面，雖有delAT1954及G>C1586+5兩個novel mutation分別在兩個不同家族被發現，但因家族數目仍佔少數，不能證明此即為台灣家族性高脂血症家族的主要突變。另外，我們找到的APOB突變，其中R3500W即存在於兩個家族，而另一個西方最常被報告的R3500Q反而完全未發現，此結果與過去師大篩檢台灣一般高血脂病患中發現R3500W的發生率遠高於R3500Q (2.4% vs 0.3%)的結果相吻合。 心血管功能分析方面，我們從18個家族中一共選出60位FH患者與另外年齡姓別配對之60位非高膽固醇血症對照組進行比較分析，在排除罹患有冠心症的患者之後，兩組之間的低密度膽固醇有顯著差異(198.8±46.8 vs 115.4±29.0 mg/dl, P<0.001)，其餘心血管危險因子並無差異，而IMT平均值在FH患者明顯比對照組增厚(62.0±14.1 vs 54.1±10.7μm, P=0.009)，FH患者也比控制組有較高比例發生心肌舒張期功能異常(29.8 vs 10.5%, OR=3.6, P=0.013)。FH患者比控制組雖然平均FMD值較低，但未達顯著差異(16.7±15.3 vs 18.3±10.1%, P=0.623)；另外，兩組之間的Tei index也沒有明顯差異(0.40±0.11 vs 0.38±0.11, P=0.567)。 總結我們的研究，台灣的家族性高膽固醇血症患者，約有六成帶有LDLR、APOB或PCSK9基因之突變，目前並無證據顯示LDLR mutation在台灣人中有founder effect；另外，台灣人的APOB突變，R3500W遠較R3500Q常見，這一點與西方人有相當大的不同。高膽固醇血症會使頸動脈內膜中層厚度顯著增厚，也代表未來罹患心血管疾病的危險性增加；另外，高膽固醇血症患者相較一般人有較高的危險性罹患心肌舒張功能異常，這也是第一個前瞻性研究證明高膽固醇血症對人體心肌舒張功\\能具有不良影響。

【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.