先天性静脉畸形骨肥大综合征和长QT综合征的分子遗传学研究
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摘要
心血管疾病是最重要的人类疾病之一,在欧美发达国家和中国都是导致人类
死亡的的头号杀手。对心血管疾病的分子遗传学研究,不仅可提供精确的诊断方
法,发展新的有效的治疗手段,同时还有助于我们理解心脏工作和血管发育的机
理。在本研究中,我们克隆了一个新的血管生成因子,VG5Q, 其突变可引起先天
性静脉畸形骨肥大综合征(Klippel-Trenaunay Syndrome,以下简称 KTS);在一
个大的长 QT 家系中,我们发现了一个新的 KCNQ1 基因的突变,L187P, 该突变引
起这一家庭的部分成员患有呈常染色体显性遗传的长 QT 综合征。
KTS 是一类先天性血管疾病,其症状是毛细血管、静脉管、淋巴管畸形,以及
骨和软组织肥大,可影响到身体的许多部位,出现疼痛、残疾和畸形,给病人带来
极大的痛苦和精神压力。KTS通常散发,其发病原因尚不清楚。
本文通过定位克隆的方法克隆了一个新基因 VG5Q,并研究了该基因在血管生成
过程中的生理功能。我们在一位发生了 t(5;11)(q13.3;p15.1)平衡易位的 KTS 病人身
上分离淋巴细胞,通过体细胞杂交,获得了含衍生 5 号染色体和衍生 11 号染色体
的人-仓鼠杂种细胞株。用 PCR 我们得到了两条染色体的精确的易位断裂点,使得
我们能鉴定其附近的 KTS 候选基因,结果在 5 号染色体上的断裂点附近,克隆了一
个在血管细胞中高度表达的基因,我们将之命名为 VG5Q.
在发生平衡易位 t(5;11)的病人中,易位断裂发生在 VG5Q 上游的启动子区或
调控区内,距 VG5Q 的转录起始点仅 1274bp,在人脐带静脉内皮细胞中,带有易位
的 11 号染色体部分 DNA 序列的 VG5Q 启动子,较野生型的表达活性提高约三倍,提
示这位 KTS 患者的染色体平衡易位是一种导致 VG5A 过度表达的遗传缺陷。
对 130 位散发的 KTS病人,我们进行了 VG5Q 的全部编码序列的突变检测,
结果发现在 5 位患者中,都发生了相同的第三外显子的第 133 位密码子的 G-A替
换,这一突变引起带负电荷的谷氨酸为带正电荷的赖氨酸所取代。对 200 位正常对
照的 SSCP 分析未能发现这一突变。
应用鸡胚尿囊绒膜检测,我们发现纯化的细菌表达的 VG5Q 蛋白能强烈地促
血管生成;与血管生成因子 VEGF 相似,在含 VG5Q 的玻璃纤维滤纸周围可见轮
状的丰富的新生血管。我们还发现野生型和 E133K 突变型 VG5Q 的促血管生成作
用存在着显著的差异,突变型蛋白具有更强的促血管生成功能。
我们认为,VG5Q 是一个 KTS 的致病基因。其根据如下:1,KTS 病人的平衡易
位发生在 VG5Q 的启动子区域,而易位可使得内皮细胞的 VG5Q 表达增强三倍。2,
在 130 位 KTS 病人的突变检测中,有五位患者具有 VG5Q 的 E133K 突变,而在 200
位正常对照中,我们未发现这一变化。3,VG5Q 在血管细胞中高度表达,而 KTS 的
最主要症状是血管畸形。4,和血管生成因子 VEGF 相同,VG5Q 可强烈促进血管生
成。5,和野生型 VG5Q 相比,E133K 突变型的 VG5Q 具有更强的促血管生成作用,表
明这一突变是一种功能增强型突变。6,在 11 号染色体的易位断裂点附近 100kb 的
范围内,尚未有基因被发现。所有这些,加上文献报道 KTS 患者病变部位的毛细血
管和静脉管增多,强有力地支持我们的结论:VG5Q 是一种新的血管生成因子,它
是第一个被鉴定的 KTS 的致病基因。
1
复旦大学博士论文 先天性静脉畸形骨肥大综合征和长 QT 综合征的分子遗传学研究
长QT综合征是指心电图上有QT间期延长、易产生室性心律失常,尤其是
尖端扭转性室速,患者易发生晕厥和猝死的一类心脏病。我们对一个具有 5 代人的
呈常染色体显性遗传的长 QT 家系进行研究,我们选用了五个与 6 种长 QT 基因紧
密连锁的微卫星标记(D11S4046、D7S798、D3S1277、D4S402 和 D21S266)进行
连锁分析,结果表明,该家系的致病基因与 LQT1 紧密连锁(Lod Score 为 6.11);
对 KCNQ1 基因全部编码区的测序和 SSCP分析, 发现病人的第 187 号密码子发生
T-C 的碱基替换,引起亮氨酸被脯氨酸取代的错义突变。SSCP和 RFLP 分析的结
果,表明在此家系中,该突变与疾病共分离,而 200 位正常对照中则未发现该突
变。
通过对该家系的基因诊断,发现在接受测试的 42 名成员中,31 位为突变基因
的携带者,其中 26 位具有典型的 LQT1 的 ECGs 模式;18 位致病基因携带者具有
正常或临界范围的 QTc(0.45±0.04 sec), 提示在这个 LQT1 家庭中,KCNQ1 基因中
的 L187P 改变是一种外显率较低的遗传突变,而基因诊断对这类疾病的预防具有
重要意义。
Cardiovascular diseases are the No.1 killer in the United States, other developed
countries and China. The molecular genetic research on cardiovascular disease will not
only lead to development of accurate genetic testing and novel therapies for
cardiovascular diseases, but also provide insights into molecular mechanisms underlying
the development and function of the heart and blood vessels. In this study, we have
cloned a novel angiogenic factor, VG5Q, as a candidate gene for Klippel-Trenaunay
Syndrome; we also identified a novel mutation L187P of KCNQ1 gene, in a five
generation family with autosomal dominant LQTS.
Klippel-Trenaunay Syndrome (OMIM *149000) is a congenital vascular disease
characterized by malformations of capillary, venous, and lymphatic vessels, with bony
and soft tissue hypertrophy. It affects many parts of the body, and is associated with
significant morbidity with a profound impact on a patient’s life (e.g., pain, disability,
disfigurement, and social stress). KTS is commonly sporadic, and its etiology is
unknown.
In this study, we used positional cloning to identify a novel gene, VG5Q, for KTS,
and explore the physiological function of VG5Q in angiogenesis. A translocation
t(5;11)(q13.3;p15.1) has previously been found to be associated with KTS. PCR analysis
with somatic cell hybrids containing only the derivative chromosome 5 or the derivative
chromosome 11 defined the precise locations of two translocation breakpoints. It allows
us to identify KTS candidate gene, and a novel gene, named VG5Q (Vascular Gene on
5q), close to the breakpoints on chromosome 5 has been identified. The 5q13.3
translocation breakpoint is located in the promoter/regulatory region of VG5Q, and is
only 1274 bp upstream from the beginning of VG5Q cDNA. VG5Q promoter with the
translocation junction fragment increased its expression by 3.0 fold in human umbilical
vein endothelial cells, suggests that the t (5; 11) translocation in the KTS patient is a
functional genetic defect that can lead to over-expression of VG5Q.
We performed mutational analysis for VG5Q with 130 single KTS patients. A
single non-conservative VG5Q mutation E133K was identified in five independent KTS
patients, and this mutation results in substitution of a negatively charged glutamine
residue for a positively charged lysine residue .Mutation E133K was not detected in 200
normal subjects.
With the chick chorioallantoic membrane (CAM) assay, we found that the purified,
wild type VG5Q protein promoted strong angiogenesis as shown by the newly formed,
radiated vessels on the CAM. Similar results were observed around the discs which were
spotted with VEGF. Marked differences in angiogenesis were observed between wild
type and mutant VG5Q with the E133K substitution. Mutant VG5Q protein produced a
significantly more potent angiogenic factor than the wild type protein.
3
复旦大学博士论文 先天性静脉畸形骨肥大综合征和长 QT 综合征的分子遗传学研究
Several lines of evidence strongly support the involvement of VG5Q in the
pathogenesis of KTS: i) The KTS translocation breakpoint is located in the promoter
region of VG5Q, and the translocation increased expression of VG5Q by 3 fold in
endothelial cells; ii) Mutation E133K of VG5Q was identified in five independent
patients, but not in 200 comparable controls; iii) VG5Q is highly expressed in blood
vessels; iv) VG5Q can cause angiogenesis (as potently as VEGF); v) Mutant VG5Q
with E133K causes greater angiogenesis than wild type VG5Q, establishing E133K as a
functional mutation that acts by a gain-of function mechanism; vi) No genes were
identified at the chromosome 11p15.1 breakpoint (100 kb region). Together with the
histological finding of greatly increased numbers of capillary veins in affected tissues
from KTS patients, these results provide strong support for increased angiogenesis as the
molecular pathogenic mechanism
死亡的的头号杀手。对心血管疾病的分子遗传学研究,不仅可提供精确的诊断方
法,发展新的有效的治疗手段,同时还有助于我们理解心脏工作和血管发育的机
理。在本研究中,我们克隆了一个新的血管生成因子,VG5Q, 其突变可引起先天
性静脉畸形骨肥大综合征(Klippel-Trenaunay Syndrome,以下简称 KTS);在一
个大的长 QT 家系中,我们发现了一个新的 KCNQ1 基因的突变,L187P, 该突变引
起这一家庭的部分成员患有呈常染色体显性遗传的长 QT 综合征。
KTS 是一类先天性血管疾病,其症状是毛细血管、静脉管、淋巴管畸形,以及
骨和软组织肥大,可影响到身体的许多部位,出现疼痛、残疾和畸形,给病人带来
极大的痛苦和精神压力。KTS通常散发,其发病原因尚不清楚。
本文通过定位克隆的方法克隆了一个新基因 VG5Q,并研究了该基因在血管生成
过程中的生理功能。我们在一位发生了 t(5;11)(q13.3;p15.1)平衡易位的 KTS 病人身
上分离淋巴细胞,通过体细胞杂交,获得了含衍生 5 号染色体和衍生 11 号染色体
的人-仓鼠杂种细胞株。用 PCR 我们得到了两条染色体的精确的易位断裂点,使得
我们能鉴定其附近的 KTS 候选基因,结果在 5 号染色体上的断裂点附近,克隆了一
个在血管细胞中高度表达的基因,我们将之命名为 VG5Q.
在发生平衡易位 t(5;11)的病人中,易位断裂发生在 VG5Q 上游的启动子区或
调控区内,距 VG5Q 的转录起始点仅 1274bp,在人脐带静脉内皮细胞中,带有易位
的 11 号染色体部分 DNA 序列的 VG5Q 启动子,较野生型的表达活性提高约三倍,提
示这位 KTS 患者的染色体平衡易位是一种导致 VG5A 过度表达的遗传缺陷。
对 130 位散发的 KTS病人,我们进行了 VG5Q 的全部编码序列的突变检测,
结果发现在 5 位患者中,都发生了相同的第三外显子的第 133 位密码子的 G-A替
换,这一突变引起带负电荷的谷氨酸为带正电荷的赖氨酸所取代。对 200 位正常对
照的 SSCP 分析未能发现这一突变。
应用鸡胚尿囊绒膜检测,我们发现纯化的细菌表达的 VG5Q 蛋白能强烈地促
血管生成;与血管生成因子 VEGF 相似,在含 VG5Q 的玻璃纤维滤纸周围可见轮
状的丰富的新生血管。我们还发现野生型和 E133K 突变型 VG5Q 的促血管生成作
用存在着显著的差异,突变型蛋白具有更强的促血管生成功能。
我们认为,VG5Q 是一个 KTS 的致病基因。其根据如下:1,KTS 病人的平衡易
位发生在 VG5Q 的启动子区域,而易位可使得内皮细胞的 VG5Q 表达增强三倍。2,
在 130 位 KTS 病人的突变检测中,有五位患者具有 VG5Q 的 E133K 突变,而在 200
位正常对照中,我们未发现这一变化。3,VG5Q 在血管细胞中高度表达,而 KTS 的
最主要症状是血管畸形。4,和血管生成因子 VEGF 相同,VG5Q 可强烈促进血管生
成。5,和野生型 VG5Q 相比,E133K 突变型的 VG5Q 具有更强的促血管生成作用,表
明这一突变是一种功能增强型突变。6,在 11 号染色体的易位断裂点附近 100kb 的
范围内,尚未有基因被发现。所有这些,加上文献报道 KTS 患者病变部位的毛细血
管和静脉管增多,强有力地支持我们的结论:VG5Q 是一种新的血管生成因子,它
是第一个被鉴定的 KTS 的致病基因。
1
复旦大学博士论文 先天性静脉畸形骨肥大综合征和长 QT 综合征的分子遗传学研究
长QT综合征是指心电图上有QT间期延长、易产生室性心律失常,尤其是
尖端扭转性室速,患者易发生晕厥和猝死的一类心脏病。我们对一个具有 5 代人的
呈常染色体显性遗传的长 QT 家系进行研究,我们选用了五个与 6 种长 QT 基因紧
密连锁的微卫星标记(D11S4046、D7S798、D3S1277、D4S402 和 D21S266)进行
连锁分析,结果表明,该家系的致病基因与 LQT1 紧密连锁(Lod Score 为 6.11);
对 KCNQ1 基因全部编码区的测序和 SSCP分析, 发现病人的第 187 号密码子发生
T-C 的碱基替换,引起亮氨酸被脯氨酸取代的错义突变。SSCP和 RFLP 分析的结
果,表明在此家系中,该突变与疾病共分离,而 200 位正常对照中则未发现该突
变。
通过对该家系的基因诊断,发现在接受测试的 42 名成员中,31 位为突变基因
的携带者,其中 26 位具有典型的 LQT1 的 ECGs 模式;18 位致病基因携带者具有
正常或临界范围的 QTc(0.45±0.04 sec), 提示在这个 LQT1 家庭中,KCNQ1 基因中
的 L187P 改变是一种外显率较低的遗传突变,而基因诊断对这类疾病的预防具有
重要意义。
Cardiovascular diseases are the No.1 killer in the United States, other developed
countries and China. The molecular genetic research on cardiovascular disease will not
only lead to development of accurate genetic testing and novel therapies for
cardiovascular diseases, but also provide insights into molecular mechanisms underlying
the development and function of the heart and blood vessels. In this study, we have
cloned a novel angiogenic factor, VG5Q, as a candidate gene for Klippel-Trenaunay
Syndrome; we also identified a novel mutation L187P of KCNQ1 gene, in a five
generation family with autosomal dominant LQTS.
Klippel-Trenaunay Syndrome (OMIM *149000) is a congenital vascular disease
characterized by malformations of capillary, venous, and lymphatic vessels, with bony
and soft tissue hypertrophy. It affects many parts of the body, and is associated with
significant morbidity with a profound impact on a patient’s life (e.g., pain, disability,
disfigurement, and social stress). KTS is commonly sporadic, and its etiology is
unknown.
In this study, we used positional cloning to identify a novel gene, VG5Q, for KTS,
and explore the physiological function of VG5Q in angiogenesis. A translocation
t(5;11)(q13.3;p15.1) has previously been found to be associated with KTS. PCR analysis
with somatic cell hybrids containing only the derivative chromosome 5 or the derivative
chromosome 11 defined the precise locations of two translocation breakpoints. It allows
us to identify KTS candidate gene, and a novel gene, named VG5Q (Vascular Gene on
5q), close to the breakpoints on chromosome 5 has been identified. The 5q13.3
translocation breakpoint is located in the promoter/regulatory region of VG5Q, and is
only 1274 bp upstream from the beginning of VG5Q cDNA. VG5Q promoter with the
translocation junction fragment increased its expression by 3.0 fold in human umbilical
vein endothelial cells, suggests that the t (5; 11) translocation in the KTS patient is a
functional genetic defect that can lead to over-expression of VG5Q.
We performed mutational analysis for VG5Q with 130 single KTS patients. A
single non-conservative VG5Q mutation E133K was identified in five independent KTS
patients, and this mutation results in substitution of a negatively charged glutamine
residue for a positively charged lysine residue .Mutation E133K was not detected in 200
normal subjects.
With the chick chorioallantoic membrane (CAM) assay, we found that the purified,
wild type VG5Q protein promoted strong angiogenesis as shown by the newly formed,
radiated vessels on the CAM. Similar results were observed around the discs which were
spotted with VEGF. Marked differences in angiogenesis were observed between wild
type and mutant VG5Q with the E133K substitution. Mutant VG5Q protein produced a
significantly more potent angiogenic factor than the wild type protein.
3
复旦大学博士论文 先天性静脉畸形骨肥大综合征和长 QT 综合征的分子遗传学研究
Several lines of evidence strongly support the involvement of VG5Q in the
pathogenesis of KTS: i) The KTS translocation breakpoint is located in the promoter
region of VG5Q, and the translocation increased expression of VG5Q by 3 fold in
endothelial cells; ii) Mutation E133K of VG5Q was identified in five independent
patients, but not in 200 comparable controls; iii) VG5Q is highly expressed in blood
vessels; iv) VG5Q can cause angiogenesis (as potently as VEGF); v) Mutant VG5Q
with E133K causes greater angiogenesis than wild type VG5Q, establishing E133K as a
functional mutation that acts by a gain-of function mechanism; vi) No genes were
identified at the chromosome 11p15.1 breakpoint (100 kb region). Together with the
histological finding of greatly increased numbers of capillary veins in affected tissues
from KTS patients, these results provide strong support for increased angiogenesis as the
molecular pathogenic mechanism
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附录:攻读博士期间发表或待发表的论文
1. Xiaoli Tian*, Mugen Liu*, Rajkumar Kadaba*, Ayse Anil Timur, David J.
Driscoll, Julian Borrow and Qing Wang, Positional cloning of a novel
angiogenic factor gene: VG5Q mutations are associated with
Klippel-Trenaunay syndrome; submitted to Nature(*并列第一作者)
2. Liu M, Zhang L, Wang L, Chen S, Vincent GM, and Wang Q. (2003)
Novel mutation L187P in KCNQ1 causes autosomal dominant long QT
syndrome. Am J Hum Genet 73 (5): 405 (Supplement).
3. Fan C, Liu M, Wang Q. Functional analysis of TBX5 missense mutations
associated with Holt-Oram syndrome. J Biol Chem. 2003 Mar 7; 278(10)
: 8780-5.
4. Liu L, Chen H, Liu M, Jin L, Wei Y, Wu X, Liu Y, Xu R, Chai J
Two novel mutations of the retinitis pigmentosa GTPase regulator gene in
two Chinese families with X-linked retinitis pigmentosa.
Chin Med J (Engl). 2002 Jun; 115(6):833-6.
5. 刘木根,赵寿元, 单核苷酸多态简述,生命科学,2000,12(6)
6. Liu L, Jin L, Liu M, Wei Y, Wu X, Liu Y, Wang H, Chu R, Chai J,
Identification of two novel mutations (E332X and c1536delC) in the RPGR
gene in two Chinese families with X-linked retinitis pigmentosa.
Hum Mutat. 2000 Jun; 15(6):584.
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23. Splawski, I.; Tristani-Firouzi, M.; Lehmann, M. H.; Sanguinetti, M. C.; Keating, M. T.
Mutations in the hminK gene cause long QT syndrome and suppress I(Ks) function.
Nature Genet. 17: 338-340, 1997.
24. Arrighi, I.; Bloch-Faure, M.; Grahammer, F et al. Altered potassium balance and
aldosterone secretion in a mouse model of human congenital long QT syndrome. Proc.
Nat. Acad. Sci. 98: 8792-8797, 2001.
25.Abbott, G. W.; Sesti, F.; Splawsky, I et al. MiRP1 forms I(kr) potassium channels
with HERG and is associated with cardiac arrhythmia. Cell 97: 175-187, 1999.
26. Moss AJ, Zareba W, Benhorin J, et al. ECG T-wave patterns in genetically distinct
forms of the hereditary long QT syndrome, Circulation 92:2929–2934, 1995.
27. Zareba W, Moss AJ, Schwartz PJ, et al. Influence of the genotype on the clinical
course of the long-QT syndrome. N Engl J Med 339:960–965, 1998.
28.Schwartz PJ, Priori SG, Locati ETT, et al. Long qt syndrome patients with mutations
of the SCN5A and HERG genes have differential responses to Na+ channel blockade and
to increases in heart rate. Circulation 92:3381–3386, 1995.
29.Fouad Y. Shalaby,Paul C. Levesque, Wen-Pin Yang, Wayne A. Little, Mary Lee
Conder, Tonya Jenkins-West, Michael A. Blanar. Dominant-Negative KvLQT1
Mutations Underlie the LQT1 Form of Long QT Syndrome. Circulation. 96:1733-1736,
1997.
30. Shimizu W, Kurita T, Matsuo K, et al. Improvement of repolarization abnormalities
by a K+ channel opener in LQTl form of congenital long-QT syndrome. Circulation
97:1581–1588, 1998.
31. Igor Splawski, Jiaxiang Shen, Katherine W. Spectrum of Mutations in Long-QT
Syndrome Genes KVLQT1, HERG, SCN5A, KCNE1, and KCNE2 .Circulation.
102:1178, 2000.
32. G. M. Vincent.The Long-QT Syndrome -- Bedside to Bench to Bedside
N. Engl. J. Med., 348(19): 1837 –1838, 2003.
93
复旦大学博士论文 先天性静脉畸形骨肥大综合征和长 QT 综合征的分子遗传学研究
附录:攻读博士期间发表或待发表的论文
1. Xiaoli Tian*, Mugen Liu*, Rajkumar Kadaba*, Ayse Anil Timur, David J.
Driscoll, Julian Borrow and Qing Wang, Positional cloning of a novel
angiogenic factor gene: VG5Q mutations are associated with
Klippel-Trenaunay syndrome; submitted to Nature(*并列第一作者)
2. Liu M, Zhang L, Wang L, Chen S, Vincent GM, and Wang Q. (2003)
Novel mutation L187P in KCNQ1 causes autosomal dominant long QT
syndrome. Am J Hum Genet 73 (5): 405 (Supplement).
3. Fan C, Liu M, Wang Q. Functional analysis of TBX5 missense mutations
associated with Holt-Oram syndrome. J Biol Chem. 2003 Mar 7; 278(10)
: 8780-5.
4. Liu L, Chen H, Liu M, Jin L, Wei Y, Wu X, Liu Y, Xu R, Chai J
Two novel mutations of the retinitis pigmentosa GTPase regulator gene in
two Chinese families with X-linked retinitis pigmentosa.
Chin Med J (Engl). 2002 Jun; 115(6):833-6.
5. 刘木根,赵寿元, 单核苷酸多态简述,生命科学,2000,12(6)
6. Liu L, Jin L, Liu M, Wei Y, Wu X, Liu Y, Wang H, Chu R, Chai J,
Identification of two novel mutations (E332X and c1536delC) in the RPGR
gene in two Chinese families with X-linked retinitis pigmentosa.
Hum Mutat. 2000 Jun; 15(6):584.
94