三种位于ADAMTS13编码去整合素样结构域上的基因突变可导致蛋白产物功能缺陷并又发血栓形成

doi:10.16150/j.1671-2870.2025.04.010

摘要/Abstract

摘要：

目的：本研究分析血管性血友病因子裂解酶 13（a disintegrin and metalloproteinase with thrombospondin motifs 13，ADAMTS13）编码去整合素样结构域处存在基因突变的血栓患者基因型和表型特征，探讨基因突变导致的血栓形成机制。方法：采用本团队自建的基于第二代测序和CNVplex®高通量拷贝数变异检测技术的中国人易栓症基因Panel（包含35个中国人群中常见的相关易栓症基因），在2020年7月至2024年8月间连续筛查1130例来我院血栓与止血门诊就诊的血栓患者，筛查出携带ADAMTS13突变的血栓患者，纳入基因突变位点仅存在于ADAMTS13编码去整合素样结构域上的先证者，并进行家系调查。对于先证者，采用凝固法检测其凝血功能，使用免疫比浊法测定血管性血友病因子（von Willebrand factor， vWF）活性和抗原水平；采用酶联免疫吸附法检测vWF的胶原结合能力；采用十二烷基硫酸-琼脂糖凝胶电泳分析患者血浆vWF不同分子量多聚体的分布情况，并进行灰度值半定量分析。采用荧光共振能量转移法测定先证者血浆中 ADAMTS13 活性，采用酶联免疫吸附法检测ADAMTS13抗原水平。使用PyMOL软件对野生型及突变型ADAMTS13的三维结构进行分析，探究突变对蛋白产物功能的影响。结果：本研究共筛查出87例携带ADAMTS13基因突变的血栓患者，其中4例先证者（4/87）携带的突变仅存在于编码去整合素样结构域处。4例易栓症先证者及相应的家系成员被纳入分析，先证者及携带相同基因突变的家系成员均经历了不同程度的血栓事件，包括脑静脉窦血栓、下肢深静脉血栓及肺栓塞。4例先证者携带的编码ADAMTS13去整合素样结构域的基因突变，均为杂合突变，分别位于8号外显子c. 901C>G（p.Pro301Ala）突变（先证者1），8号外显子c.902C>G（p.Pro301Arg）（先证者2、3），9号外显子c.1045C>T（p.Arg 349Cys）（先证者4），经查询人类基因突变数据库（Human Gene Mutation Database，HGMD），其中 p.Pro301Ala 突变、p.Pro301Arg 突变尚无登记。凝血功能检测表明，4 例患者 ADAMTS13 活性（Act 为 57.42%~72.88%）和抗原水平（Ag 为 66.94%~78.34%）显著降低，vWF活性（Act为158.2%~213.7%）和抗原水平（Ag为167.2%~216.6%）升高。vWF多聚体电泳分析显示，患者血浆中高分子量多聚体（high-molecular-weight multimers， HMWMs）比例显著增加，提示HMWMs较正常人明显增多，灰度值为166.6~218.9比117.4。PyMOL软件分析，提示突变位点位于ADAMTS13编码去整合素样结构域的关键区域，可导致ADAMTS13蛋白的稳定性下降，与vWF的结合能力下降。结论：本研究首次报道位于ADAMTS13编码去整合素样结构域上的2个新突变（p.Pro301Ala和p.Pro301Arg），这2个突变与另一例p.Arg349Cys（已报道）一样，被证实可导致ADAMTS13抗原水平及蛋白活性下降，表现为ADAMTS13对vWF高分子量多聚体的裂解能力减弱，患者体内存在异常的vWF多聚体比例上升，ADAMTS13-vWF轴的动态平衡被破坏，患者血栓形成的风险上升。

关键词: 血管性血友病因子裂解酶13, 血管性血友病因子, 去整合素样结构域, 基因突变, 血栓形成

Abstract:

Objective This study aims to investigate the mechanisms of thrombosis caused by gene mutations by analyzing the genotypic and phenotypic characteristics of thrombotic patients with mutations in the disintegrin-like domain encoded by a disintegrin and metalloproteinase with thrombospondin 1 motifs 13 (ADAMTS13). Methods A thrombophilia gene panel for the Chinese population was established, which was based on next-generation sequencing and CNVplex® high-throughput copy number variation detection technology and included 35 thrombophilia-related genes commonly found in the Chinese population. Between July 2020 and August 2024, a total of 1 130 thrombotic patients attending the Thrombosis and Hemostasis Clinic at our hospital were continuously screened using this panel. Patients carrying ADAMTS13 mutations were identified, and probands with mutation sites located exclusively within the disintegrin-like domain encoded by ADAMTS13 were included for family investigation. For the probands, the coagulation function was evaluated using clotting tests. The activity and antigen levels of von Willebrand factor (vWF) were measured by immunoturbidimetry. The collagen-binding capacity of vWF was assessed using enzyme-linked immunosorbent assay. The distribution of vWF multimers with different molecular weights in patient plasma was analyzed using sodium dodecyl sulfate-agarose gel electrophoresis, followed by semi-quantitative grayscale value analysis. The activity of ADAMTS13 in the probands’ plasma was measured using the fluorescence resonance energy transfer method, and ADAMTS13 antigen levels were determined using enzyme-linked immunosorbent assay. The three-dimensional structures of wild-type and mutant ADAMTS13 were analyzed using PyMOL software to investigate the impact of the mutations on protein function. Results A total of 87 thrombotic patients carrying ADAMTS13 gene mutations were identified, among whom four probands (4/87) had mutations located exclusively within the disintegrin-like domain. These four probands with thrombophilia and their corresponding family members were included in the analysis. Both the probands and the family members carrying the same gene mutations experienced thrombotic events of varying severity, including cerebral venous sinus thrombosis, deep vein thrombosis of the lower extremities, and pulmonary embolism. All four probands carried heterozygous mutations in the gene encoding the disintegrin-like domain of ADAMTS13, which were located in exon 8 c. 901C>G (p. Pro301Ala) in proband 1, exon 8 c. 902C>G (p.Pro301Arg) in probands 2 and 3, and exon 9 c. 1045C>T (p. Arg349Cys) in proband 4. Verification with the Human Gene Mutation Database (HGMD) confirmed that the p. Pro301Ala and p. Pro301Arg mutations had not been previously registered. Coagulation function testing revealed that ADAMTS13 activity (Act: 57.42%-72.88%) and antigen levels (Ag: 66.94%-78.34%) were significantly reduced in all four patients, while vWF activity (Act: 15.2%-213.7%) and antigen levels (Ag: 167.2%-216.6%) were markedly increased. Electrophoretic analysis of vWF multimers showed that the proportion of high-molecular-weight multimers (HMWMs) in the patients’ plasma was significantly increased, indicating a marked elevation of HMWMs compared to normal individuals, with grayscale values ranging from 166. 6 to 218. 9 versus 117. 4. Analysis using PyMOL software indicated that the mutation sites were located in key regions of the disintegrin-like domain encoded by ADAMTS13, which may reduce the stability of the ADAMTS13 protein and decrease its binding capacity to vWF. Conclusions This study reports, for the first time, two novel mutations (p. Pro301Ala and p. Pro301Arg) in the disintegrin-like domain encoded by ADAMTS13. Similar to the previously reported p. Arg349Cys mutation, these two mutations have been confirmed to reduce the antigen levels and activity of the ADAMTS13 protein. This is manifested as impaired cleavage capacity of ADAMTS13 towards HMWMs of vWF, resulting in an abnormal increase in the proportion of vWF multimers, disruption of the dynamic balance of the ADAMTS13- vWF axis, and an elevated risk of thrombosis in these patients.

Key words: ADAMTS13, von Willebrand factor, Disintegrin-like domain, Gene mutation, Thrombosis

中图分类号:

R554
R446.11

林莉亚, 吴希, 毛胤祺, 陈光明, 武文漫, 戴菁, 王学锋, 丁秋兰. 三种位于ADAMTS13编码去整合素样结构域上的基因突变可导致蛋白产物功能缺陷并又发血栓形成[J]. 诊断学理论与实践, 2025, 24(04): 431-440.

LIN Liya, WU Xi, MAO Yinqi, CHEN Guangming, WU Wenman, DAI Jing, WANG Xuefeng, DING Qiulan. Three gene mutations in disintegrin-like domain encoded by ADAMTS13 causing functional defects in protein products and inducing thrombosis[J]. Journal of Diagnostics Concepts & Practice, 2025, 24(04): 431-440.

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Proband	Gene Analysis	ADAMTS13: Act（%）	ADAMTS13: Ag（%）	vWF:Act （%）	vWF:Ag （%）	vWF:CB /vWF:Ag（Ratio）	ADAMTS13:Act /vWF:Ag（Ratio）
1	ADAMTS13 c.901C>G:p.Pro301Ala*	62.03	70.35	211.6	214.9	1.47	0.289
2	ADAMTS13 c.902C>G:p.Pro301Arg*	67.35	75.46	213.7	216.6	1.28	0.311
3	ADAMTS13 c.902C>G:p.Pro301Arg*	72.88	78.34	158.2	167.2	1.39	0.436
4	ADAMTS13 c.1045C>T:p.Arg349Cys	57.42	66.94	179.5	188.9	1.33	0.304
	Normal range	60.00-150.00	40.00-120.00	50.0-150.0	50.0-150.0	0.70-1.20	0.500-2.000