中国人群eNOS基因多态性与妊高征相关性研究
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摘要
妊娠高血压综合征（PIH）是严重危害孕产妇和待产儿健康的疾病之一，但
是其发病机制至今仍未弄清楚。NO是体内最强的血管舒张因子，目前普遍认为内
源性NO产量的减少可能是导致妊高征发生的重要原因之一。体内满足生理功能
所需要的NO主要由内皮型一氧化氮合酶（eNOS）催化L-精氨酸生成瓜氨酸与NO
的反应提供，并且eNOS是该反应途径中唯一的限速酶。妊高征患者体内NO合成
量的减少可能是由于eNOS基因突变所致。故人们推测eNOS基因可能是妊高征的
易感基因。位于eNOS基因外显子7上G894T突变导致其编码蛋白298位发生
Glu/Asp的错义突变，该突变存在影响eNOS蛋白的稳定性，从而降低蛋白的催化
活性。另外位于eNOS基因启动子上T-786C突变能显著降低该启动子的转录活性，
从而减少eNOS蛋白的合成，NO合成量也随之减少。为证实这两个多态性是否同
样在中国人群中存在以及频率分布情况，并判定是否是妊高征发病的风险因子。
我们以中国人群（包括妊高征患者组、正常妊娠组、随机妇女组及随机人群组）
为研究对象，分别用PCR-RFLP和PCR-SSCP与测序和PCR-TGGE的方法检测了eNOS
基因G894T和T-786C这两个多态性位点的存在并统计各自的频率分布。结果在
51例中、重度妊高征患者和56例正常妊娠者、74例随机妇女以及82例随机人
群中外显子7突变基因型（GT，TT）频率分别为35.4％、31.1％、26.8％、27.5％
（P=0.751），T基因频率分别为18.3％、15.5％、13.4％、13.7％（P=0.661）；同时
在55例中、重度妊高征患者和82例正常妊娠以及96例随机妇女中启动子突变
基因型（CC，TC）频率分别为19.8％、15.9％、20％（P=0.951），C等位基因的频
率分别为10.9％、9.0％、11.8％（P=0.755）。因此，在中国人群中存在外显子7 G894T
和启动子T-786C这两个多态性位点。应用病例-对照分析的方法分析了这两个多
态性与中、重度妊高征的相关性。结果发现这两个多态性位点均与中、重度妊高
征不相关。我们未能找到eNOS基因是妊高征的候选基因的直接证据。eNOS基因
是否是妊高征的易感基因还需进一步实验证明。
Pregnancy induced hypertension (PIH) is one of severe
diseases which badly endanger the health of pregnancy woman
and fetus. Genetic factors appear to be important in
pathogenesis of PIH, but the molecular basis of this genetic
predisposition remains largely unknown. Vascular endothelial
cells produce nitric oxide (NO), a vascular smooth muscle
relaxing factor which plays an important role in the regulation
of blood pressure and regional blood flow. NO is produced by
endothelial nitric oxide synthase (eNOS) from L梐rginine, eNOS
is only key synthase in this pathway. A large number of studies
have shown that decreasing of NO is contributed to development
of PIH. The mutation of eNOS gene resulted in decreasing of NO
produce within PIH woman. So eNOS gene may be the candidate gene
for PIH. A variant within exon 7 of eNOS gene: G to T conversion
at nucleotide position 894 resulting in replacement of glutainic
acid aspartic acide at condon 298 effects the stability of eNOS
protein and results in decreasing of NO produce. Another variant
within promoter of eNOS gene: T to C conversion at nucleotide
position ?86 results in reducing promoter activity by ~5O%
and impairs NO biosynthesis. To explore this two variants of
eNOS gene, and to analysis the relationship between the eNOS
3
gene and PIH women in Chinese population, polymerase chain
reaction 梤estriction fragment length polymorphism (PCR桼FLP)
analysis was used to screen the variant G8941 in exon7 of eNOS
gene in PIH and normotensive pregnancy women and random women
and random people subjects; polymerase chain reaction single
strand conformation polymorphism (PCR桽SCP) with PCR
direct梥equencing analysis and polymerase chain reaction
梩emperature gradient gel electophersis (PCR桾GGE) analysis
were used to screen the variant T?86C in promoter of eNOS gene
in PIH and normotensive pregnancy women and random women
subjects. The frequencies of variants (GT, TT) of exon 7 on 51
middle, server PIH women and 56 normotensive pregnancy women and
74 random women and 82 random peoples subjects were 35. 4%, 31. 1%,
26. 8%, 27. 5%, respectively. There were no statistical different
in gene type of variants (GG, TI) of exon 7 among them (P0. 751).
The I allele frequencies of exon 7 in middle, server PIH was
18. 3%, in normotensive pregnancy women was 15. 5%, in random
women was 13. 4%, and in random peoples was 13. 7%. There were
also no statistical different in I allele of eNOS gene exon7
among them (P0. 661). Another, the frequencies of variants (IC,
CC) of promoter on 55 middle, server PJH women and 82
normotensive pregnancy women and 96 random women subjects were
19.8%, 15.9%, 20%, respectively. There were no statistical
different in gene type of variants (IC, CC) of promoter of eNOS
gene among them (P0. 951). The C allele frequencies of promoter
in middle, server PIH was 10. 9%, in normotensive pregnancy women
was 9.0%, and in random women was 11.8%. There were also no
statistical different in C allele of promoter of eNOS gene among
them (P0. 775). The results do not suggest that G894T in exon7
and I?86C in promoter of the eNOS gene are involved in
development of PIH. Further work must to be done to seek evidence
4
for association be
是其发病机制至今仍未弄清楚。NO是体内最强的血管舒张因子，目前普遍认为内
源性NO产量的减少可能是导致妊高征发生的重要原因之一。体内满足生理功能
所需要的NO主要由内皮型一氧化氮合酶（eNOS）催化L-精氨酸生成瓜氨酸与NO
的反应提供，并且eNOS是该反应途径中唯一的限速酶。妊高征患者体内NO合成
量的减少可能是由于eNOS基因突变所致。故人们推测eNOS基因可能是妊高征的
易感基因。位于eNOS基因外显子7上G894T突变导致其编码蛋白298位发生
Glu/Asp的错义突变，该突变存在影响eNOS蛋白的稳定性，从而降低蛋白的催化
活性。另外位于eNOS基因启动子上T-786C突变能显著降低该启动子的转录活性，
从而减少eNOS蛋白的合成，NO合成量也随之减少。为证实这两个多态性是否同
样在中国人群中存在以及频率分布情况，并判定是否是妊高征发病的风险因子。
我们以中国人群（包括妊高征患者组、正常妊娠组、随机妇女组及随机人群组）
为研究对象，分别用PCR-RFLP和PCR-SSCP与测序和PCR-TGGE的方法检测了eNOS
基因G894T和T-786C这两个多态性位点的存在并统计各自的频率分布。结果在
51例中、重度妊高征患者和56例正常妊娠者、74例随机妇女以及82例随机人
群中外显子7突变基因型（GT，TT）频率分别为35.4％、31.1％、26.8％、27.5％
（P=0.751），T基因频率分别为18.3％、15.5％、13.4％、13.7％（P=0.661）；同时
在55例中、重度妊高征患者和82例正常妊娠以及96例随机妇女中启动子突变
基因型（CC，TC）频率分别为19.8％、15.9％、20％（P=0.951），C等位基因的频
率分别为10.9％、9.0％、11.8％（P=0.755）。因此，在中国人群中存在外显子7 G894T
和启动子T-786C这两个多态性位点。应用病例-对照分析的方法分析了这两个多
态性与中、重度妊高征的相关性。结果发现这两个多态性位点均与中、重度妊高
征不相关。我们未能找到eNOS基因是妊高征的候选基因的直接证据。eNOS基因
是否是妊高征的易感基因还需进一步实验证明。
Pregnancy induced hypertension (PIH) is one of severe
diseases which badly endanger the health of pregnancy woman
and fetus. Genetic factors appear to be important in
pathogenesis of PIH, but the molecular basis of this genetic
predisposition remains largely unknown. Vascular endothelial
cells produce nitric oxide (NO), a vascular smooth muscle
relaxing factor which plays an important role in the regulation
of blood pressure and regional blood flow. NO is produced by
endothelial nitric oxide synthase (eNOS) from L梐rginine, eNOS
is only key synthase in this pathway. A large number of studies
have shown that decreasing of NO is contributed to development
of PIH. The mutation of eNOS gene resulted in decreasing of NO
produce within PIH woman. So eNOS gene may be the candidate gene
for PIH. A variant within exon 7 of eNOS gene: G to T conversion
at nucleotide position 894 resulting in replacement of glutainic
acid aspartic acide at condon 298 effects the stability of eNOS
protein and results in decreasing of NO produce. Another variant
within promoter of eNOS gene: T to C conversion at nucleotide
position ?86 results in reducing promoter activity by ~5O%
and impairs NO biosynthesis. To explore this two variants of
eNOS gene, and to analysis the relationship between the eNOS
3
gene and PIH women in Chinese population, polymerase chain
reaction 梤estriction fragment length polymorphism (PCR桼FLP)
analysis was used to screen the variant G8941 in exon7 of eNOS
gene in PIH and normotensive pregnancy women and random women
and random people subjects; polymerase chain reaction single
strand conformation polymorphism (PCR桽SCP) with PCR
direct梥equencing analysis and polymerase chain reaction
梩emperature gradient gel electophersis (PCR桾GGE) analysis
were used to screen the variant T?86C in promoter of eNOS gene
in PIH and normotensive pregnancy women and random women
subjects. The frequencies of variants (GT, TT) of exon 7 on 51
middle, server PIH women and 56 normotensive pregnancy women and
74 random women and 82 random peoples subjects were 35. 4%, 31. 1%,
26. 8%, 27. 5%, respectively. There were no statistical different
in gene type of variants (GG, TI) of exon 7 among them (P0. 751).
The I allele frequencies of exon 7 in middle, server PIH was
18. 3%, in normotensive pregnancy women was 15. 5%, in random
women was 13. 4%, and in random peoples was 13. 7%. There were
also no statistical different in I allele of eNOS gene exon7
among them (P0. 661). Another, the frequencies of variants (IC,
CC) of promoter on 55 middle, server PJH women and 82
normotensive pregnancy women and 96 random women subjects were
19.8%, 15.9%, 20%, respectively. There were no statistical
different in gene type of variants (IC, CC) of promoter of eNOS
gene among them (P0. 951). The C allele frequencies of promoter
in middle, server PIH was 10. 9%, in normotensive pregnancy women
was 9.0%, and in random women was 11.8%. There were also no
statistical different in C allele of promoter of eNOS gene among
them (P0. 775). The results do not suggest that G894T in exon7
and I?86C in promoter of the eNOS gene are involved in
development of PIH. Further work must to be done to seek evidence
4
for association be
引文
1． Rodgers GM, Taylor RN, Roberts JM. Preeclampsia is associated with a serum factor cytotoxic to human endothelial cells. Am J Obstet Gynecol, 1988, 159 :908-914
2． 汪希鹏,林其德.妊高征的遗传研究.中华妇产科杂志, 1998, 33:
3． Brenner B, Lanir N, Thaler I. HELLP syndrome associated with factor V R506Q mutation. Br J Haematol, 1996, 92:999-1001
4． Chen G, Wilson R, Wang SH et al. Tumor necrosis factor-alpha (TNF-α ) gene polymorphism and expression in pre-eclampsia. Clin Exp Immunol, 1996, 104:154-159
5． Arnggrimsson R, Hayward C, Nadaud S, et al. Evidence for a familial pregnancy-induced hypertension locus in the eNOS gene region. Am J Hum Genet,1997, 61:354-362
6． Arngrimsson R, Purandare S, Connor M. Angiotensinogen: a candiate gene involed in preeclampsia. Nat Genet, 1993, 4:114-115
7． Ward K, Hata A, Jeunemaitre X, et al. A molecular variant of angiotensinogen associated with preeclampsia. Nat Genet, 1993, 4:59-61
8． Kobashi G. A case-control study of pregnancy induced hypertension with a genetic predisposition: association of a molecular variant of angiotensinogen in the Japanese woman Hokkaido. Lgaku Zasshi, 1995, 70:649-657
9． Inoue I, Rohrwasser A, Helin C, et al. A mutation of angiotensinogen in a patient with preeclampsia leads to altered kinetics of the renin-angiotension system. J Biol Chem, 1995, 270:11430-11436
10． Morgan L, Baker P, Pipken FB, et al. Preeclampsia and the angiotensinogen gene. Br J Obstet Gynecol, 1995, 102:489-490
11． Zeeman GG, Dekker GA, Vangeijin HP, et al. Endothelial function in normal and preeclampsia pregnancy : a hypothesis. Eur J Obstet Gynecol Reprod Biot, 1992, 43:113-122
12． Seligman SP, Bayon JP, Clancy RM, et al. The role of nitric oxide in the pathogenesis of preeclmpsia. Am J Obstet Gynecol, 1994, 171:944-948
13． 周天鸿,李月琴,姚冬生。妊高征易感基因研究进展。遗传, 2000, 22 (4) : 262-264
14． Molner M, Suto T, Toth T, et al. Prolonged blockade of nitric oxide synthesis in gravid rats produces sustained hypertension, proteinurin, thrombrocytopenia, and intrauterine growth retardation. Am J Obstet Gynecol, 1996, 103:879-886
15． Lubarsky SL, Ahokas RA, Friedam SA. The effect of chronic nitric oxide synthesis inhibition on blood pressure and angiotensis Ⅱ responsiveness in the pregnancy rat. Am J Obstet Gynecol, 1997, 176:1069-1076
16． Marsden PA, Heng HH, Scherer SW, et al. Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. J Biol Chem , 1993, 268(23) :17478-88
17． Karantzoulis-Fegras F, Antoniou H, Lai SL, et al. Characterization of the human endothelial nitric-oxide synthase promoter. J Biol Chem, 1999, 274(5) :3076-3093
18． Nakayama T, Soma M, Takayashi Y, et al. Association analysis of CA repeat polymorphism of the endothelial nitric oxide synthase gene with essential hypertension in Japanese. Clin Genet, 1997, 51:26-30
19． Bonnardeaux AS, Nadaud S, Charru A, et al. Lack of evidence for linkage of the cell nitric oxide synthase gene to essential hypertension. Circulation, 1995, 91:96-102
20． Hunt SC, Williams CS, Sharma AM, et al. Lack of linkage between the endothelial nitric oxide synthase gene and hypertension. J Hum Hypertens, 1996, 10:27-30
21． Wang XL, Sim AS, Badenhop RF, et al. A smoking-dependent risk of coronary artery disease associated with a polymorphism of the endothelial nitric oxide synthase gene. Nat Med, 1996, 2:41-45
22． Uwabo J, Soma M, Nakayama T, et al. Association of a variable number
of tandem repeats in the endothelial constitutive nitric oxide synthase gene with essential hypertension in Japanese. Am J Hypertens, 1998, 11:125-128
23． Tesauro M, Thompson WC, Rogliani P, et al. Intracellular processing of endothelial nitric oxide synthase isoforms associated with difference in severity of cardiopulmonary diseases: cleavage of proteins with aspartate vs. glutamate at position 298． Proc Natl Acad Sci USA, 2000, 97 (6) :2832-2835
24． Shimasaki Y, Yasue H, Yoshimura M, et al. Association of the missense Glu298Asp variant of the endothelial nitric oxide synthase gene with myocardial infarction. JACC, 1998, 31 (7) :1506-1510
25． Liao YL, Saku K, Ou FJ, et al. A missense mutation of the nitric oxide synthase (eNOS) gene (Glu298Asp) in five patients with coronary artery disease. Angiology, 1999, 50(8) :671-676
26． Miyamoto Y, Saito Y, Kajiyama N, et al. Endothelial nitric oxide synthase gene is positively association with essential hypertension.
27． Shoji M, Tsutaya S, Saito R et al. Positive association of endothelial nitric oxide synthase gene polymorphism with hypertension in northern Japan. Life Science, 2000, 66(26) :2557-2562
28． Schneider MP, Erdmann J, Delles C, et al. Functional gene testing of the Glu298Asp polymorphism of the endothelial NO synthase. J Hypertens, 2000 , 18(12) :1767-73．
29． Nakayama M, Yasue H, Yoshimura M, et al. T~(-786) C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation, 1999, 99:2864-2870
30． Thomas F, Luscher, Georg N. Is it all in the gene...? Nitric oxide synthase and coronary vasospasm. Circulation, 1999, 99:2855-2857
31． Zanchi A, Moczulski DK, Hanna LS, et al. Risk of advanced diabetic nephropathy in type 1 diabetes is associated with endothelial nitric oxide synthase gene polymorphism. Kidney International, 2000, 57:405-413
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43. Sladek SM, Magness RR, Conrad KP. Nitric oxide and pregnancy. Am J Physiol, 1997, 272:R441-R463
44. Baylis C, Beinder E, S(?)t(?) T, et al. Recent insights into the roles of nitric oxide and renin-angiotensin in the pathophysiology of preeclamptic pregnancy. Seminars in nephrology, 1998, 18(2):208-230
45． Neri I, Renzo GCD, Caserta G, et al. Impact of the L-arginine/nitric oxide system in pregnancy. Obstetrical and Gynecological Survey, 1995, 50(12) :851-858
46． Morris NH, Eaton BM, Dekker G. Nitric oxide , the endothelium, pregnancy and pre-eclampsia. Br J Obstet Gynaecol , 1996, 103:4-15
47． Markus Hs, Ruigrok Y, Ali N, et al. endothelial nitric oxide synthase exon7 polymorphism, ischemic cerebrovascular disease, and carotid atheroma. Stroke, 1998, setember: 1908-1911．
48． Kato N, sugiyama T, Morita H, et al. Lack of evidence for association between the endothelia nitric oxide synthase gene and hypertension. Hypertension, 1999, 33:933-936．
49． Macleod MJ, Dahiyat MT, Cumming A, et al. No association between Glu/Asp polymorphism of NOS3 gene and ischemic stroke. Neurology, 1999, 53:418-420．
50． Philip I, PlantefeveG, Vuillaumier-Barrot S, et al. G894T polymorphism in the endothelial nitric oxide synthase gene is associated with an enhanced vascular responsiveness to phenylephrine. Circulation, 1999, 99(24) :3096-3098
51． Zhou R, Li W, Cao Z. The changes and significance of the intensity of transcription of endothelial nitric oxide synthase-mRNA in placental tissue from cases of pregnancy induced hypertension. Zhonghua Fu Chan Ke Za Zhi, 1998, 33(12) : 709-10
2． 汪希鹏,林其德.妊高征的遗传研究.中华妇产科杂志, 1998, 33:
3． Brenner B, Lanir N, Thaler I. HELLP syndrome associated with factor V R506Q mutation. Br J Haematol, 1996, 92:999-1001
4． Chen G, Wilson R, Wang SH et al. Tumor necrosis factor-alpha (TNF-α ) gene polymorphism and expression in pre-eclampsia. Clin Exp Immunol, 1996, 104:154-159
5． Arnggrimsson R, Hayward C, Nadaud S, et al. Evidence for a familial pregnancy-induced hypertension locus in the eNOS gene region. Am J Hum Genet,1997, 61:354-362
6． Arngrimsson R, Purandare S, Connor M. Angiotensinogen: a candiate gene involed in preeclampsia. Nat Genet, 1993, 4:114-115
7． Ward K, Hata A, Jeunemaitre X, et al. A molecular variant of angiotensinogen associated with preeclampsia. Nat Genet, 1993, 4:59-61
8． Kobashi G. A case-control study of pregnancy induced hypertension with a genetic predisposition: association of a molecular variant of angiotensinogen in the Japanese woman Hokkaido. Lgaku Zasshi, 1995, 70:649-657
9． Inoue I, Rohrwasser A, Helin C, et al. A mutation of angiotensinogen in a patient with preeclampsia leads to altered kinetics of the renin-angiotension system. J Biol Chem, 1995, 270:11430-11436
10． Morgan L, Baker P, Pipken FB, et al. Preeclampsia and the angiotensinogen gene. Br J Obstet Gynecol, 1995, 102:489-490
11． Zeeman GG, Dekker GA, Vangeijin HP, et al. Endothelial function in normal and preeclampsia pregnancy : a hypothesis. Eur J Obstet Gynecol Reprod Biot, 1992, 43:113-122
12． Seligman SP, Bayon JP, Clancy RM, et al. The role of nitric oxide in the pathogenesis of preeclmpsia. Am J Obstet Gynecol, 1994, 171:944-948
13． 周天鸿,李月琴,姚冬生。妊高征易感基因研究进展。遗传, 2000, 22 (4) : 262-264
14． Molner M, Suto T, Toth T, et al. Prolonged blockade of nitric oxide synthesis in gravid rats produces sustained hypertension, proteinurin, thrombrocytopenia, and intrauterine growth retardation. Am J Obstet Gynecol, 1996, 103:879-886
15． Lubarsky SL, Ahokas RA, Friedam SA. The effect of chronic nitric oxide synthesis inhibition on blood pressure and angiotensis Ⅱ responsiveness in the pregnancy rat. Am J Obstet Gynecol, 1997, 176:1069-1076
16． Marsden PA, Heng HH, Scherer SW, et al. Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. J Biol Chem , 1993, 268(23) :17478-88
17． Karantzoulis-Fegras F, Antoniou H, Lai SL, et al. Characterization of the human endothelial nitric-oxide synthase promoter. J Biol Chem, 1999, 274(5) :3076-3093
18． Nakayama T, Soma M, Takayashi Y, et al. Association analysis of CA repeat polymorphism of the endothelial nitric oxide synthase gene with essential hypertension in Japanese. Clin Genet, 1997, 51:26-30
19． Bonnardeaux AS, Nadaud S, Charru A, et al. Lack of evidence for linkage of the cell nitric oxide synthase gene to essential hypertension. Circulation, 1995, 91:96-102
20． Hunt SC, Williams CS, Sharma AM, et al. Lack of linkage between the endothelial nitric oxide synthase gene and hypertension. J Hum Hypertens, 1996, 10:27-30
21． Wang XL, Sim AS, Badenhop RF, et al. A smoking-dependent risk of coronary artery disease associated with a polymorphism of the endothelial nitric oxide synthase gene. Nat Med, 1996, 2:41-45
22． Uwabo J, Soma M, Nakayama T, et al. Association of a variable number
of tandem repeats in the endothelial constitutive nitric oxide synthase gene with essential hypertension in Japanese. Am J Hypertens, 1998, 11:125-128
23． Tesauro M, Thompson WC, Rogliani P, et al. Intracellular processing of endothelial nitric oxide synthase isoforms associated with difference in severity of cardiopulmonary diseases: cleavage of proteins with aspartate vs. glutamate at position 298． Proc Natl Acad Sci USA, 2000, 97 (6) :2832-2835
24． Shimasaki Y, Yasue H, Yoshimura M, et al. Association of the missense Glu298Asp variant of the endothelial nitric oxide synthase gene with myocardial infarction. JACC, 1998, 31 (7) :1506-1510
25． Liao YL, Saku K, Ou FJ, et al. A missense mutation of the nitric oxide synthase (eNOS) gene (Glu298Asp) in five patients with coronary artery disease. Angiology, 1999, 50(8) :671-676
26． Miyamoto Y, Saito Y, Kajiyama N, et al. Endothelial nitric oxide synthase gene is positively association with essential hypertension.
27． Shoji M, Tsutaya S, Saito R et al. Positive association of endothelial nitric oxide synthase gene polymorphism with hypertension in northern Japan. Life Science, 2000, 66(26) :2557-2562
28． Schneider MP, Erdmann J, Delles C, et al. Functional gene testing of the Glu298Asp polymorphism of the endothelial NO synthase. J Hypertens, 2000 , 18(12) :1767-73．
29． Nakayama M, Yasue H, Yoshimura M, et al. T~(-786) C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation, 1999, 99:2864-2870
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