2型糖尿病家系中高甘油三酯血症患者脂蛋白酯酶基因突变及功能分析
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摘要
1.2 型糖尿病家系中高甘油三酯血症患者脂蛋白脂酶基因分子筛查
目的:对成都地区汉族人群2型糖尿病(T2DM)家系中伴高甘油三酯血症(HTG)患者进行脂蛋白脂酶(LPL)基因分子筛查。
方法:选取成都地区汉族人群中符合入选条件的T2DM家系,从T2DM家系中选择TG≥2.25mmol/L的患者进行LPL基因分子筛查,53人符合此标准,其中T2DM患者31人,糖耐量减低者(IGT)11人,糖耐量正常(NGT)者11人,这53人共代表了26个T2DM家系,TG范围在2.3—13.0mmol/L。正常对照118例。用聚合酶链反应—单链构象多态性(PCR—SSCP)和聚合酶链反应—变性高效液相色谱(PCR—DHPLC)对LPL基因进行突变检测,然后对检出的突变位点通过DNA序列分析进一步证实,对发现突变位点的家系成员进行筛查,同时在正常群体中筛查这些突变位点的频率。对所有研究对象均进行血糖、血脂和胰岛素测定,计算体重指数(BMI)。
结果:1.在LPL基因外显子检测到7种突变,分别是:Ala71 Thr、Vall08Val、Leu286Pro、Asn291Ser、Lys312insC、Thr361insA和Leu376Leu。其中Lys312insC、Thr361insA和Leu376Leu是本研究中新发现的突变位点,以前从未报道过。2.对新发现的Lys312insC和Thr361insA突变位点在家系成员中进行进一步筛查,结果显示8#
1. Molecular screening of the lipoprotein Iipase gene in hypertriglyceridemic members of type 2 diabetic pedigreesAim: To investigate the lipoprotein lipase(LPL) gene mutations in hypertriglyceridemic members of type 2 diabetic pedigrees in a Chinese population in Chengdu.Methods: Members of type 2 diabetic pedigrees were chosen for analysis of LPL gene if fasting triglyceride(TG) levels exceeded 2.25mmol/L. Fifty-three individuals met the criteria, including 31 individuals with type 2 diabetes mellitus(T2DM), 11 individuals with impaired glucose tolerance(IGT), and 11 individuals with normal glucose tolerance(NGT). These subjects represented 26 type 2 diabetic pedigrees. TG levels ranged from 2.3mmol/L to 13.0mmol/L. The control population consisted of 118 healthy individuals chosen from non-diabetic families. Polymerase chain reaction(PCR)-single strand conformation polymorphism (SSCP), PCR-denaturing high performance liquid chromatography(DHPLC) and direct DNA sequencing were applied for analysis of LPL gene.
Results: 1. A total of 7 different LPL variants were detected among 53 individuals of type 2 diabetic pedigrees. Four of the variants were previously detected in exon 3 (Ala71Thr and VaI108Val), and exon 6 (Leu286Pro and Asn291Ser). Three novel variants were detected in exon6 (Lys312 insertion C) and in exon 8 (Thr361 insertion A and Leu376Leu). 2. The LPL gene in the family members of the novel Lys312insC and Thr361insA mutations pedigrees were further analyzed. In 8# family, 6 individuals who carried Lys312insC mutation had a high triglyceridemia, and three of them also carried the Asn291Ser mutation had a much higher triglyceridemia, so maybe there was a strong linkage disequilibrium between the Lys312insC mutation and the Asn291Ser mutation; In 14# and 28# family, the Thr361insA mutation cosegregated with the phenotype (hypertriglyceridemia) and was thus suggested to have a pathophysiological effect; No individual carried with Lys312insC and Thr361insA mutations in control group. 3. In new 1# pedigree, the proband whose TG was 11.9mmol/L was found carrying the Leu286Pro mutation; and in new 3# pedigree, the proband whose TG was 13.0mmol/L was found carrying the Ala71Thr mutation. The mutation, Leu286Pro and Ala71Thr, reported previously, had a significant association with hypertriglyceridemia, so we think the mutation contribute to the hypertriglyceridemia in this two pedigrees.Conclusion: The genetic variants at the LPL gene occur
commonly in hypertriglyceridemic members of type 2 diabetic pedigrees in a Chinese population in Chengdu. LPL variants contribute to the hypertriglyceridemia in some families.
2. Construction, clone and identification of recombinantwild lipoprotein lipase gene plasmidAim: To obtain the recombinant wild lipoprotein lipase(LPL) gene plasmid.Methods: The total RNA was isolated from the human epiploon adipose tissue. According to the reported cDNA sequence of the LPL, primers were designed and synthesized. By means of RT-PCR,the LPL cDNA was obtained. The LPL cDNA was cloned into the pcDNA3.1Zeo (+) vector. The recombinant pcDNA3.1Zeo (+) /LPL cDNA was identified by endonucleases, PCR and DNA sequencing.Results: The LPL gene had been cloned into the pcDNA3.1Zeo (+) plasmid identified by endonucleases and PCR. The sequence of the LPL gene was the same to the sequence of the Gene Bank identified by DNA sequencing.Conclusion: The recombinant plasmid pcDNA3.1Zeo (+)/LPL cDNA was successfully constructed.
3. Site-directed mutation and expression in vitro of recombinant LPL gene plasmidAim: To obtain the recombinant mutant LPL gene plasmid and to investigate the function of the mutant LPL gene.Methods: Mutant LPL cDNAs with the desired mutations were generated from the pcDNA3.1Zeo(+)-wild type LPL cDNA using the Site-directed Kit (Takara, Japan). COS-1 cells were transfecteed with the recombinant LPL gene plasmid using LIPOFECTAMINE 2000?(LF2000, Gibco BRL, Life Technologies, USA). The LPL mass in cells and the culture medium were determined by a Markit-M LPL Kit (Dainippon Pharmaceutical Co., Ltd, Japan). Spectrophotometry was used to measure the LPL activity.Results: 1. Mutant pcDNA3.1Zeo(+)/LPLcDNA plasmids with the mutations Asn291Ser, Lys312insC, Thr361insA and Asn291 Ser + Lys312insC were generated and transformed into the COS-1 cells. 2. LPL mass and enzyme activity in both culture medium and cell lysate were measured. COS-1 cells and pcDNA3.1Zeo(+) plasmid themselves expressed no LPL. The COS-1 cells expressed LPL mass and activity when transfected with wild type LPL cDNA. All mutations transfected COS-1 cells produced significantly decreased LPL activities in both cell lysates and medium. The LPL activities were undetectable in cell lysates and medium of the Asn291Ser+Lys312insC transfected cells. The LPL mass of all mutant types was also significantly lower in culture medium compared to the mass of the wild
type, but all mutant types had LPL mass similar to that of the wild type in cell lysates.Conclusion: the mutations Asn291Ser, Lys312insC, Thr361insA and Asn291Ser+Lys312insC did not influence the transcription and translation of LPL, but affected the secretion and activity of the enzyme to various degrees.
目的:对成都地区汉族人群2型糖尿病(T2DM)家系中伴高甘油三酯血症(HTG)患者进行脂蛋白脂酶(LPL)基因分子筛查。
方法:选取成都地区汉族人群中符合入选条件的T2DM家系,从T2DM家系中选择TG≥2.25mmol/L的患者进行LPL基因分子筛查,53人符合此标准,其中T2DM患者31人,糖耐量减低者(IGT)11人,糖耐量正常(NGT)者11人,这53人共代表了26个T2DM家系,TG范围在2.3—13.0mmol/L。正常对照118例。用聚合酶链反应—单链构象多态性(PCR—SSCP)和聚合酶链反应—变性高效液相色谱(PCR—DHPLC)对LPL基因进行突变检测,然后对检出的突变位点通过DNA序列分析进一步证实,对发现突变位点的家系成员进行筛查,同时在正常群体中筛查这些突变位点的频率。对所有研究对象均进行血糖、血脂和胰岛素测定,计算体重指数(BMI)。
结果:1.在LPL基因外显子检测到7种突变,分别是:Ala71 Thr、Vall08Val、Leu286Pro、Asn291Ser、Lys312insC、Thr361insA和Leu376Leu。其中Lys312insC、Thr361insA和Leu376Leu是本研究中新发现的突变位点,以前从未报道过。2.对新发现的Lys312insC和Thr361insA突变位点在家系成员中进行进一步筛查,结果显示8#
1. Molecular screening of the lipoprotein Iipase gene in hypertriglyceridemic members of type 2 diabetic pedigreesAim: To investigate the lipoprotein lipase(LPL) gene mutations in hypertriglyceridemic members of type 2 diabetic pedigrees in a Chinese population in Chengdu.Methods: Members of type 2 diabetic pedigrees were chosen for analysis of LPL gene if fasting triglyceride(TG) levels exceeded 2.25mmol/L. Fifty-three individuals met the criteria, including 31 individuals with type 2 diabetes mellitus(T2DM), 11 individuals with impaired glucose tolerance(IGT), and 11 individuals with normal glucose tolerance(NGT). These subjects represented 26 type 2 diabetic pedigrees. TG levels ranged from 2.3mmol/L to 13.0mmol/L. The control population consisted of 118 healthy individuals chosen from non-diabetic families. Polymerase chain reaction(PCR)-single strand conformation polymorphism (SSCP), PCR-denaturing high performance liquid chromatography(DHPLC) and direct DNA sequencing were applied for analysis of LPL gene.
Results: 1. A total of 7 different LPL variants were detected among 53 individuals of type 2 diabetic pedigrees. Four of the variants were previously detected in exon 3 (Ala71Thr and VaI108Val), and exon 6 (Leu286Pro and Asn291Ser). Three novel variants were detected in exon6 (Lys312 insertion C) and in exon 8 (Thr361 insertion A and Leu376Leu). 2. The LPL gene in the family members of the novel Lys312insC and Thr361insA mutations pedigrees were further analyzed. In 8# family, 6 individuals who carried Lys312insC mutation had a high triglyceridemia, and three of them also carried the Asn291Ser mutation had a much higher triglyceridemia, so maybe there was a strong linkage disequilibrium between the Lys312insC mutation and the Asn291Ser mutation; In 14# and 28# family, the Thr361insA mutation cosegregated with the phenotype (hypertriglyceridemia) and was thus suggested to have a pathophysiological effect; No individual carried with Lys312insC and Thr361insA mutations in control group. 3. In new 1# pedigree, the proband whose TG was 11.9mmol/L was found carrying the Leu286Pro mutation; and in new 3# pedigree, the proband whose TG was 13.0mmol/L was found carrying the Ala71Thr mutation. The mutation, Leu286Pro and Ala71Thr, reported previously, had a significant association with hypertriglyceridemia, so we think the mutation contribute to the hypertriglyceridemia in this two pedigrees.Conclusion: The genetic variants at the LPL gene occur
commonly in hypertriglyceridemic members of type 2 diabetic pedigrees in a Chinese population in Chengdu. LPL variants contribute to the hypertriglyceridemia in some families.
2. Construction, clone and identification of recombinantwild lipoprotein lipase gene plasmidAim: To obtain the recombinant wild lipoprotein lipase(LPL) gene plasmid.Methods: The total RNA was isolated from the human epiploon adipose tissue. According to the reported cDNA sequence of the LPL, primers were designed and synthesized. By means of RT-PCR,the LPL cDNA was obtained. The LPL cDNA was cloned into the pcDNA3.1Zeo (+) vector. The recombinant pcDNA3.1Zeo (+) /LPL cDNA was identified by endonucleases, PCR and DNA sequencing.Results: The LPL gene had been cloned into the pcDNA3.1Zeo (+) plasmid identified by endonucleases and PCR. The sequence of the LPL gene was the same to the sequence of the Gene Bank identified by DNA sequencing.Conclusion: The recombinant plasmid pcDNA3.1Zeo (+)/LPL cDNA was successfully constructed.
3. Site-directed mutation and expression in vitro of recombinant LPL gene plasmidAim: To obtain the recombinant mutant LPL gene plasmid and to investigate the function of the mutant LPL gene.Methods: Mutant LPL cDNAs with the desired mutations were generated from the pcDNA3.1Zeo(+)-wild type LPL cDNA using the Site-directed Kit (Takara, Japan). COS-1 cells were transfecteed with the recombinant LPL gene plasmid using LIPOFECTAMINE 2000?(LF2000, Gibco BRL, Life Technologies, USA). The LPL mass in cells and the culture medium were determined by a Markit-M LPL Kit (Dainippon Pharmaceutical Co., Ltd, Japan). Spectrophotometry was used to measure the LPL activity.Results: 1. Mutant pcDNA3.1Zeo(+)/LPLcDNA plasmids with the mutations Asn291Ser, Lys312insC, Thr361insA and Asn291 Ser + Lys312insC were generated and transformed into the COS-1 cells. 2. LPL mass and enzyme activity in both culture medium and cell lysate were measured. COS-1 cells and pcDNA3.1Zeo(+) plasmid themselves expressed no LPL. The COS-1 cells expressed LPL mass and activity when transfected with wild type LPL cDNA. All mutations transfected COS-1 cells produced significantly decreased LPL activities in both cell lysates and medium. The LPL activities were undetectable in cell lysates and medium of the Asn291Ser+Lys312insC transfected cells. The LPL mass of all mutant types was also significantly lower in culture medium compared to the mass of the wild
type, but all mutant types had LPL mass similar to that of the wild type in cell lysates.Conclusion: the mutations Asn291Ser, Lys312insC, Thr361insA and Asn291Ser+Lys312insC did not influence the transcription and translation of LPL, but affected the secretion and activity of the enzyme to various degrees.
引文
1. Goldberg U, Merkel M. Lipoprotein lipase: physiology, biochemistry, and molecular biology. Frontiers in Bioscience, 2001,6(3): d388-405.
2. Deeb S, Peng R. Structure of the human lipoprotein lipase gene. Biochemistry, 1989,28(11):4131-4135.
3. Winston AH, Lawrence C, Wen-Hsiung L. Structure and evolution of the lipase superfamily. J Lipid Res, 1992, 33(2): 167-178
4. Olivecrona T, Hernell O, Egelrud T. Lipoprotein lipase. Adv Exp Med Biol, 1975;52:269-79..
5. Jap TS, Jenq SF, Wu YC, et al. Mutations in the lipoprotein lipase gene as a cause of hypertriglyceridemia and pancreatitis in taiwan. Pancreas,2003,27(2): 122-126.
6. Benlian P, De Gennes JL, Foubert L, et al. Premature atherosclerosis in patients with familial chylomicronemia caused by mutations in the lipoprotein lipase gene. N Engl J Med,1996, 335(12):848-854.
7. Wilson DE, Hata A, Kwong LK, et al. Mutation in exon 3 of the lipoprotein lipase gene segregating in a family with hypertriglyceridemia, pancreatitis, and non-insulin-dependent diabetes. J Clin Invest,1993,92(1):203-211.
8. Chadarevian R, Foubert L, Beucler I, et al. Lipoprotein lipase activity and common gene variants in severely hypertriglyceridemic patients with and without diabetes. Horm Res, 2003, 60(1): 61-67.
9. Geltrude MFL, Henriksen AV, Greco LN, et al. Triglyceride-induced diabetes associated with familial lipoprotein lipase deficiency. Diabetes, 1999,48(6): 1258-1263.
10. Tao Yang, Chi-Pui Pang, Man-Wo Tsang, et al. Pathogenic mutations of the lipoprotein lipase gene in chinese patients with hypertriglyceridemic type 2 diabetes. HUMAN MUTATION Mutation in Brief #605 (2003) Online
11.惠汝太,周宪梁主编。心血管分子生物学实验技术。北京:北京医科大学中国协和医科大学联合出版社,1998年。
12.马立人,王立全主编。真核细胞染色体DNA的制备。基因诊断研究新进展。北京:人民军医出版社,1997年。
13. Lander ES, Botstein D. Strategies for studying heterogeneous genetic traits in humans by using a linkage map of restriction fragment length polymorphisms. Proc Natl Acad Sci USA,1996, 83(19):7353-7357.
14. Wenz HM, Baumhueter S, Ramachandra S, et al. A rapid automated SSCP multiplex capillary electrophoresis protocol that detects the two common mutations implicated in hereditary hemochromatosis. Hum Gene, 1999,104(1):29-35.
15. Kulin A, Munson K, Gjerde D, et al. Detection of single nucleotide polymorphisms with the WAVETM DNA fragment analysis system. Genetic Testing, 1998,4:201-206.
16. Oefner PJ, Underhill PA. Comparative DNA sequencing by denaturing high-performance liquid chromatograph. Am J Hum Genet, 1995, 57: A266.
17. Benlian P, De Gennes JL, Foubert L, et al. Premature athero- sclerosis in patients with familial chylomicronemia caused by mutations in the lipoprotein lipase gene. N Engl J Med,1996, 335(12):848-854.
18. Elbein SC, Yeager C, Kwong LK, et al. Molecular screening of the lipoprotein lipase gene in hypertriglyceridemic members of familial noninsulin-dependent diabetes mellitus families. J Clin Endocrinol Metab, 1994, 79(5): 1450-1456.
19. Razzaghi H, Aston CE, Hamman RF, et al. Genetic screening of the lipoprotein lipase gene for mutations associated with high triglyceride/low HDL-cholesterol levels. Hum Genet, 2000, 107(3): 257-267.
20. Zhang Q, Cavallero E, Hoffmann MM, et al. Mutations at the lipoprotein lipase gene locus in subjects with diabetes mellitus, obesity and lipemia. Clin Sci(Colch), 1997,93 (4):335-341.
21. Pimstone SN, Clee SM, Gagne SE, et al. A frequently occurring mutation in the lipoprotein lipase gene (Asn291Ser) results in altered postprandial chylomicron triglyceride and retinyl palmitate response in normolipidaemic carriers. J Lipid Res, 1996,37(8): 1675-1684.
22.张维铭主编。现代分子生物学实验手册。北京:科学出版社,2003年。
2. Deeb S, Peng R. Structure of the human lipoprotein lipase gene. Biochemistry, 1989,28(11):4131-4135.
3. Winston AH, Lawrence C, Wen-Hsiung L. Structure and evolution of the lipase superfamily. J Lipid Res, 1992, 33(2): 167-178
4. Olivecrona T, Hernell O, Egelrud T. Lipoprotein lipase. Adv Exp Med Biol, 1975;52:269-79..
5. Jap TS, Jenq SF, Wu YC, et al. Mutations in the lipoprotein lipase gene as a cause of hypertriglyceridemia and pancreatitis in taiwan. Pancreas,2003,27(2): 122-126.
6. Benlian P, De Gennes JL, Foubert L, et al. Premature atherosclerosis in patients with familial chylomicronemia caused by mutations in the lipoprotein lipase gene. N Engl J Med,1996, 335(12):848-854.
7. Wilson DE, Hata A, Kwong LK, et al. Mutation in exon 3 of the lipoprotein lipase gene segregating in a family with hypertriglyceridemia, pancreatitis, and non-insulin-dependent diabetes. J Clin Invest,1993,92(1):203-211.
8. Chadarevian R, Foubert L, Beucler I, et al. Lipoprotein lipase activity and common gene variants in severely hypertriglyceridemic patients with and without diabetes. Horm Res, 2003, 60(1): 61-67.
9. Geltrude MFL, Henriksen AV, Greco LN, et al. Triglyceride-induced diabetes associated with familial lipoprotein lipase deficiency. Diabetes, 1999,48(6): 1258-1263.
10. Tao Yang, Chi-Pui Pang, Man-Wo Tsang, et al. Pathogenic mutations of the lipoprotein lipase gene in chinese patients with hypertriglyceridemic type 2 diabetes. HUMAN MUTATION Mutation in Brief #605 (2003) Online
11.惠汝太,周宪梁主编。心血管分子生物学实验技术。北京:北京医科大学中国协和医科大学联合出版社,1998年。
12.马立人,王立全主编。真核细胞染色体DNA的制备。基因诊断研究新进展。北京:人民军医出版社,1997年。
13. Lander ES, Botstein D. Strategies for studying heterogeneous genetic traits in humans by using a linkage map of restriction fragment length polymorphisms. Proc Natl Acad Sci USA,1996, 83(19):7353-7357.
14. Wenz HM, Baumhueter S, Ramachandra S, et al. A rapid automated SSCP multiplex capillary electrophoresis protocol that detects the two common mutations implicated in hereditary hemochromatosis. Hum Gene, 1999,104(1):29-35.
15. Kulin A, Munson K, Gjerde D, et al. Detection of single nucleotide polymorphisms with the WAVETM DNA fragment analysis system. Genetic Testing, 1998,4:201-206.
16. Oefner PJ, Underhill PA. Comparative DNA sequencing by denaturing high-performance liquid chromatograph. Am J Hum Genet, 1995, 57: A266.
17. Benlian P, De Gennes JL, Foubert L, et al. Premature athero- sclerosis in patients with familial chylomicronemia caused by mutations in the lipoprotein lipase gene. N Engl J Med,1996, 335(12):848-854.
18. Elbein SC, Yeager C, Kwong LK, et al. Molecular screening of the lipoprotein lipase gene in hypertriglyceridemic members of familial noninsulin-dependent diabetes mellitus families. J Clin Endocrinol Metab, 1994, 79(5): 1450-1456.
19. Razzaghi H, Aston CE, Hamman RF, et al. Genetic screening of the lipoprotein lipase gene for mutations associated with high triglyceride/low HDL-cholesterol levels. Hum Genet, 2000, 107(3): 257-267.
20. Zhang Q, Cavallero E, Hoffmann MM, et al. Mutations at the lipoprotein lipase gene locus in subjects with diabetes mellitus, obesity and lipemia. Clin Sci(Colch), 1997,93 (4):335-341.
21. Pimstone SN, Clee SM, Gagne SE, et al. A frequently occurring mutation in the lipoprotein lipase gene (Asn291Ser) results in altered postprandial chylomicron triglyceride and retinyl palmitate response in normolipidaemic carriers. J Lipid Res, 1996,37(8): 1675-1684.
22.张维铭主编。现代分子生物学实验手册。北京:科学出版社,2003年。