脂蛋白脂酶常见基因变异与2型糖尿病的相关研究
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摘要
背景：2型糖尿病是一种多因素疾病，虽然具有明显的遗传背景，但致病基因大部分尚未明确。2型糖尿病和其它胰岛素抵抗疾病常伴有脂质代谢异常，其特点是乳糜微粒(CM)和极低密度脂蛋白(VLDL)增高，高密度脂蛋白(HDL)降低。而且，2型糖尿病与肥胖、动脉粥样硬化、冠心病和高血压关系密切，显示出脂质代谢异常在该疾病的发生发展中起重要作用。脂蛋白脂酶(LPL)是脂质代谢的限速酶，在脂蛋白代谢中起关键作用。它能催化乳糜微粒(CM)及极低密度脂蛋白(VLDL)核心中的甘油三酯(TG)水解，促进CM和VLDL从血液循环中的清除。同时在富含TG的脂蛋白表面成分(磷脂和载脂蛋白)转变为HDL的过程中起作用，升高血浆HDL的水平。研究发现，2型糖尿病患者脂肪组织LPL的活性和含量降低。糖尿病的发病与LPL活性下降及高甘油三酯血症的发展密切相关。因此，LPL基因被认为是2型糖尿病的重要候选基因，但该基因在2型糖尿病中的变异位点和类型不明。大量国外研究表明，位于LPL基因外显子中的Asp9Asn、Asn291Ser和Ser447Ter突变能够改变LPL活性，是影响血浆TG、VLDL及HDL水平的重要因素，与高脂血症、冠心病与动脉粥样硬化的发病密切相关。其中Asp9Asn和Asn291Ser突变使LPL活性下降，对血脂代谢有负面影响，而Ser447Ter突变使LPL活性升高，产生了有益的血脂谱改变(TG降低，HDL升高)。但迄今为止，这三种基因变异在2型糖尿病中的作用尚未见报道。因此，本研究就其是否与2型糖尿病发病及患者血脂代谢紊乱相关进行探讨。
方法：研究对象由来自郑州大学第一附属医院的102名2型糖尿病患者和113名对照者组成，均为河南汉族人。两组人群在性别和年龄上无显著性差异。抽取患者和对照者空腹12h静脉血，EDTA抗凝，采用经典的SDS-蛋白酶K-酚-氯仿等方法提取基因组DNA。PCR扩增LPL基因特定片段，2％琼脂糖凝胶电泳，用pBR322 DNA／BsuRI(HaeⅢ)为分子量标准，核定PCR扩增产物的大小和扩增
郑州大学2001级硕士研究生毕业论文
脂蛋白脂酶常见基因变异与2型糖尿病的相关研究
特异性。然后分别用限制性内切酶Taql，Rsal和Hinfl进行酶切反应。最后，
进行6一10%的非变性聚丙烯酞胺凝胶电泳，分析酶切产物。所有统计学分析都
在sPSSn.o软件包中完成。用x’检验检测观察到的基因型是否符合遗传平衡。
正态分布的各统计指标都以均数士标准差(了土S)表示。病例组和对照组间血
脂及载脂蛋白水平的比较及不同基因型血脂及载脂蛋白的比较采用t检验。病例
组和对照组间基因型频率和等位基因频率的差异用x’检验。以a二0.05作为显著
性水准。
结果:LPL基因AspgAsn及Asn291ser突变在病例组和对照组中未发现，而
Ser447Ter突变位点中，病例组G等位基因和CG基因型频率均低于对照组的50%
(3.4%vs 7.1%:6.9%vs 24.2%)，但差异无统计学意义(P二0.065和P二0.057)。
病例组中，Tc和LDL均高于对照组，HDL和A训一B则低于对照组;CG基因
型携带者血浆TG水平低于非携带者，差异有显著性(P=0.032)。对照组中CG
基因型携带者血浆TG水平低于非携带者，HDL高于非携带者，差别均有统计
学意义(尸二0.012，P二0.018)。
结论:我国汉族人群脂蛋白脂酶基因Aspg户ksn、Asn291ser突变位点与2型糖尿
病无明显关联，而Ser447Tel位点突变对降低2型糖尿病发病而言可能是一个保
护性突变。病例组中ser447Ter突变携带者血浆TG水平显著低于非携带者，这
可从基因角度解释2型糖尿病患者中有些人较易出现高甘油三酷血症而另一些
人表现为血脂正常。丸pgAsn、户‘n291ser突变位点的分布可能存在种族差异。
Objective: Type 2 diabetes mellitus (Type 2 DM) is a multifactory disease. Even
though the manifestation of Type 2 DM clearly has a genetic component, the etiology of this is largely unknown. In Type 2 DM and other insulin-resistant conditions, postprandial hyperglyceridaemia and low HDL are important lipid metabolic perturbations. Moreover, a strong correlation between Type 2 DM and adiposity ^ CAD> hypertension indicats contribution from a dyslipidemic state. This implies that genes involved in lipid metabolism might be relevant candidates of a common disorder. Lipoprotein lipase (LPL) is a rate-limiting enzyme in lipid metabolism, which plays a key role in lipoprotein metabolism by catalyzing the hydrolysis of triglycerides of very low density lipoprotein (VLDL) and chylomicrons (CM), and the gene coding for LPL is therefore a candidate gene in Type 2 DM. Recently, a number of more common coding mutations in the LPL have been described, which found to influence plasma lipid. Two of these, Asp9Asn and Asn291Ser, have been described to have small deleterious effects on plasma high-dens
ity lipoprotein(HDL) cholesterol and TG, and another, Ser447Ter is reported by some investigators to underlie higher HDL cholesterol levels and lower TG , thus would represent a beneficial genetic variant in lipoprotein metabolism. However, little is know about their effects on the plasma lipoprotein metabolism of Type 2 DM. The purpose of the present study is to unravel the association between the three common gene mutations in the LPL gene and the lipid metabolic perturbations in Type 2 DM and to explore the relationship between the three common gene mutations in the LPL gene and Type 2 DM and it' s
complications.
Methods: The study population consisted of 102 patients of Type 2 DM who had been admitted to the first affiliated hospital of Zhengzhou University and 113 control subjects. All subjects enrolled in the study were Chinese Han nationality and except other endocrine disease, treatment with lipid-lowing or betablocking agents. There were no significant different in sex and age between the two groups. Subjects were drawn 3ml blood before breakfast and the genome DNA were extracted by classic SDS-proteinase K-hydroxybenzene-chloroform methods from the blood. Through PCR amplification, electrophoresis on 2% agarose gel, and EB-staining, we observed and analysed the specific segments of Asp9Asn> Asn291Ser and Ser447Ter using DNA Marker pBR322 DNA/Ztei/RI(HaeIII). And then, the segments went on being digested with restriction endonuclear enzyme Taq I, Rsa I and Hinf-I respectively. Finally, the digested segments were analysed through 6%~ 10% non-denaturation polyacrylamide gel.
All the analyses were performed with the SPSS 11.0 statistical analysis package. A chi-square test was performed to assess whether the observed genotype frequencies were in Hardy-Weinberg equilibrium. Data on age and lipid levels were analyzed by the unpaired Student' s t test. The frequencies of the alleles and the relations of genotype between the study groups were analyzed by constructing 2*2 and 2*3 contingency tables followed by chi-square analysis. A P value < 0.05 was considered significant.
Results: Type 2 DM patients had significantly lower HDL and higher TG> LDL> ApoB and Lp(a) than controls. Asp9Asn and Asn291Ser variants were not found either in 102 NIDDM patients or in 113 controls. As for the Ser447Ter mutation, the frequency of the Ter447 allele and Ser447Ter genotype were lower in cases than controls. The differences were very close to the significant level (P =0.065 and P =0.057). In control group, there were trend to lower TG and higher HDL in carriers of the Ser447Ter mutation. In NIDDM patients group, the level of plasma TG was also obviously lower in carriers of the Ser447Ter mutation than in non-carriers (P=0.032). Furthermore, the beneficial effects of Ser447Ter mutation on plasma lipids could be
seen in either sex. In the three genetic variants of LPL, the frequency of A allel in the Asp9Asn mutation and G allele in the
方法：研究对象由来自郑州大学第一附属医院的102名2型糖尿病患者和113名对照者组成，均为河南汉族人。两组人群在性别和年龄上无显著性差异。抽取患者和对照者空腹12h静脉血，EDTA抗凝，采用经典的SDS-蛋白酶K-酚-氯仿等方法提取基因组DNA。PCR扩增LPL基因特定片段，2％琼脂糖凝胶电泳，用pBR322 DNA／BsuRI(HaeⅢ)为分子量标准，核定PCR扩增产物的大小和扩增
郑州大学2001级硕士研究生毕业论文
脂蛋白脂酶常见基因变异与2型糖尿病的相关研究
特异性。然后分别用限制性内切酶Taql，Rsal和Hinfl进行酶切反应。最后，
进行6一10%的非变性聚丙烯酞胺凝胶电泳，分析酶切产物。所有统计学分析都
在sPSSn.o软件包中完成。用x’检验检测观察到的基因型是否符合遗传平衡。
正态分布的各统计指标都以均数士标准差(了土S)表示。病例组和对照组间血
脂及载脂蛋白水平的比较及不同基因型血脂及载脂蛋白的比较采用t检验。病例
组和对照组间基因型频率和等位基因频率的差异用x’检验。以a二0.05作为显著
性水准。
结果:LPL基因AspgAsn及Asn291ser突变在病例组和对照组中未发现，而
Ser447Ter突变位点中，病例组G等位基因和CG基因型频率均低于对照组的50%
(3.4%vs 7.1%:6.9%vs 24.2%)，但差异无统计学意义(P二0.065和P二0.057)。
病例组中，Tc和LDL均高于对照组，HDL和A训一B则低于对照组;CG基因
型携带者血浆TG水平低于非携带者，差异有显著性(P=0.032)。对照组中CG
基因型携带者血浆TG水平低于非携带者，HDL高于非携带者，差别均有统计
学意义(尸二0.012，P二0.018)。
结论:我国汉族人群脂蛋白脂酶基因Aspg户ksn、Asn291ser突变位点与2型糖尿
病无明显关联，而Ser447Tel位点突变对降低2型糖尿病发病而言可能是一个保
护性突变。病例组中ser447Ter突变携带者血浆TG水平显著低于非携带者，这
可从基因角度解释2型糖尿病患者中有些人较易出现高甘油三酷血症而另一些
人表现为血脂正常。丸pgAsn、户‘n291ser突变位点的分布可能存在种族差异。
Objective: Type 2 diabetes mellitus (Type 2 DM) is a multifactory disease. Even
though the manifestation of Type 2 DM clearly has a genetic component, the etiology of this is largely unknown. In Type 2 DM and other insulin-resistant conditions, postprandial hyperglyceridaemia and low HDL are important lipid metabolic perturbations. Moreover, a strong correlation between Type 2 DM and adiposity ^ CAD> hypertension indicats contribution from a dyslipidemic state. This implies that genes involved in lipid metabolism might be relevant candidates of a common disorder. Lipoprotein lipase (LPL) is a rate-limiting enzyme in lipid metabolism, which plays a key role in lipoprotein metabolism by catalyzing the hydrolysis of triglycerides of very low density lipoprotein (VLDL) and chylomicrons (CM), and the gene coding for LPL is therefore a candidate gene in Type 2 DM. Recently, a number of more common coding mutations in the LPL have been described, which found to influence plasma lipid. Two of these, Asp9Asn and Asn291Ser, have been described to have small deleterious effects on plasma high-dens
ity lipoprotein(HDL) cholesterol and TG, and another, Ser447Ter is reported by some investigators to underlie higher HDL cholesterol levels and lower TG , thus would represent a beneficial genetic variant in lipoprotein metabolism. However, little is know about their effects on the plasma lipoprotein metabolism of Type 2 DM. The purpose of the present study is to unravel the association between the three common gene mutations in the LPL gene and the lipid metabolic perturbations in Type 2 DM and to explore the relationship between the three common gene mutations in the LPL gene and Type 2 DM and it' s
complications.
Methods: The study population consisted of 102 patients of Type 2 DM who had been admitted to the first affiliated hospital of Zhengzhou University and 113 control subjects. All subjects enrolled in the study were Chinese Han nationality and except other endocrine disease, treatment with lipid-lowing or betablocking agents. There were no significant different in sex and age between the two groups. Subjects were drawn 3ml blood before breakfast and the genome DNA were extracted by classic SDS-proteinase K-hydroxybenzene-chloroform methods from the blood. Through PCR amplification, electrophoresis on 2% agarose gel, and EB-staining, we observed and analysed the specific segments of Asp9Asn> Asn291Ser and Ser447Ter using DNA Marker pBR322 DNA/Ztei/RI(HaeIII). And then, the segments went on being digested with restriction endonuclear enzyme Taq I, Rsa I and Hinf-I respectively. Finally, the digested segments were analysed through 6%~ 10% non-denaturation polyacrylamide gel.
All the analyses were performed with the SPSS 11.0 statistical analysis package. A chi-square test was performed to assess whether the observed genotype frequencies were in Hardy-Weinberg equilibrium. Data on age and lipid levels were analyzed by the unpaired Student' s t test. The frequencies of the alleles and the relations of genotype between the study groups were analyzed by constructing 2*2 and 2*3 contingency tables followed by chi-square analysis. A P value < 0.05 was considered significant.
Results: Type 2 DM patients had significantly lower HDL and higher TG> LDL> ApoB and Lp(a) than controls. Asp9Asn and Asn291Ser variants were not found either in 102 NIDDM patients or in 113 controls. As for the Ser447Ter mutation, the frequency of the Ter447 allele and Ser447Ter genotype were lower in cases than controls. The differences were very close to the significant level (P =0.065 and P =0.057). In control group, there were trend to lower TG and higher HDL in carriers of the Ser447Ter mutation. In NIDDM patients group, the level of plasma TG was also obviously lower in carriers of the Ser447Ter mutation than in non-carriers (P=0.032). Furthermore, the beneficial effects of Ser447Ter mutation on plasma lipids could be
seen in either sex. In the three genetic variants of LPL, the frequency of A allel in the Asp9Asn mutation and G allele in the
引文
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2. Jan W. Eriksson, Jonas Buren, Maria Svensson, etal. Postprandial regulation of blood lipid and adipose tissue lipoprotein lipase in type 2 diabetes patients and healthy control subjects. Arteriosclerosis, 2001; 166: 359-367
3. RB Simsolo, JM Ong, B Saffari, and PA Kern et al. Effect of improved diabetes control on the expression of lipoprotein lipase in human adipose tissue. J Lipid Res. 1992; 33 (1): 89-95
4．沈洪兵，俞顺章，徐耀初等．2型糖尿病患者脂蛋白脂酶基因Pvu Ⅱ酶切多态性的观察．中华医学遗传学杂志，2000，17(1)：24-27
5．朱宏达，孙荣，高梅等．NIDDM中脂蛋白脂酶基因多态性的研究．中华内分泌代谢杂志，1997，13(2)：73-75
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7. Steve E. Humphries, Viviane Nicaud, Joaquim Margalef, et al. Lipoprotein Lipase Gene Variation Is Associated With a Paternal History of Premature Coronary Artery Disease and Fasting and Postprandial Plasma Triglycerides. Arteriosclerosis, Thrombosis, and Vascular Biology, 1998, 18: 526-534.
8. Zhang H., P.W.A. Reymer, M.S. Liu, etal. Patients with apoE3 deficiency (E2/2, E3/2, and E4/2) who manifest with hyperlipidemia have increased frequency of an Asn 291→Set mutation in the human LPL gene. Arterioscler. Thromb. Vasc. Biol. 1995, 15: 1695-1703
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37．张秋萍，刘秉文，刘宇等．中国人内源性高甘油三酯血症患者脂蛋白脂酶及肝脂酶常见基因变异的研究．中华医学遗传学杂志，1997，14(5)：289-293
38. Zhang, H., P.W.A. Reymer, M.-S. Liu, I.J. Forsythe, B.E. Groenemeyer, J. Frohlich, J.D. Brunzell, J.J.P. Kastelein, M.R. Hayden, etal. Patients with apoE3 deficiency (E2/2, E3/2, and E4/2) who manifest with hyperlipidemia have increased frequency of an Asn 291→Ser mutation in the human LPL gene. Arterioscler. Thromb. Vasc. Biol. 1995, 15: 1695-1703
2. Jan W. Eriksson, Jonas Buren, Maria Svensson, etal. Postprandial regulation of blood lipid and adipose tissue lipoprotein lipase in type 2 diabetes patients and healthy control subjects. Arteriosclerosis, 2001; 166: 359-367
3. RB Simsolo, JM Ong, B Saffari, and PA Kern et al. Effect of improved diabetes control on the expression of lipoprotein lipase in human adipose tissue. J Lipid Res. 1992; 33 (1): 89-95
4．沈洪兵，俞顺章，徐耀初等．2型糖尿病患者脂蛋白脂酶基因Pvu Ⅱ酶切多态性的观察．中华医学遗传学杂志，2000，17(1)：24-27
5．朱宏达，孙荣，高梅等．NIDDM中脂蛋白脂酶基因多态性的研究．中华内分泌代谢杂志，1997，13(2)：73-75
6. Mailly F, Tugrul Y, Reymer PWA, et al. A Common Variant in the Gene for Lipoprotein Lipase(Asp9→Asn). Arteriosclerosis, Thrombosis, and Vascular Biology, 1995, 15: 468-478
7. Steve E. Humphries, Viviane Nicaud, Joaquim Margalef, et al. Lipoprotein Lipase Gene Variation Is Associated With a Paternal History of Premature Coronary Artery Disease and Fasting and Postprandial Plasma Triglycerides. Arteriosclerosis, Thrombosis, and Vascular Biology, 1998, 18: 526-534.
8. Zhang H., P.W.A. Reymer, M.S. Liu, etal. Patients with apoE3 deficiency (E2/2, E3/2, and E4/2) who manifest with hyperlipidemia have increased frequency of an Asn 291→Set mutation in the human LPL gene. Arterioscler. Thromb. Vasc. Biol. 1995, 15: 1695-1703
9. Tavanger K et al. J Clin Invest, 1992; 90 (5): 1672
10. ata A, Robertson M, Emi M, et al. Direct detection and automated sequencing of individual alles after electrophoretic strand separation: identification of a common nonsense mutation in exon 9 of the human lipoprotein lipasegene. Nucleic Acids Res, 1990, 18: 5407-5410
11. Galton DJ, Mattu RK, Cavanan J, et al. Polymorphism of the lipoprotein lipasegene and premature atherosclerosis. J Intern Med, 1994, 236: 63-68
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