Journal of Diagnostics Concepts & Practice >
Three gene mutations in disintegrin-like domain encoded by ADAMTS13 causing functional defects in protein products and inducing thrombosis
Received date: 2025-01-20
Revised date: 2025-02-25
Accepted date: 2025-03-25
Online published: 2025-08-25
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
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 . DOI: 10.16150/j.1671-2870.2025.04.010
| [1] | LEVY G G, NICHOLS W C, LIAN E C, et al. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura[J]. Nature, 2001, 413(6855):488-494. |
| [2] | SADLER J E. Biochemistry and genetics of von Wille-brand factor[J]. Annu Rev Biochem, 1998, 67:395-424. |
| [3] | DE GROOT R, BARDHAN A, RAMROOP N, et al. Essential role of the disintegrin-like domain in ADAMTS13 function[J]. Blood, 2009, 113(22):5609-5616. |
| [4] | FENG Y, LI X Y, XIAO J, et al. ADAMTS13: more than a regulator of thrombosis[J]. Int J Hematol, 2016, 104(5):534-539. |
| [5] | ZHENG X, CHUNG D, TAKAYAMA T K, et al. Structure of von Willebrand factor-cleaving protease (ADAMTS13), a metalloprotease involved in thrombotic thrombocytopenic purpura[J]. J Biol Chem, 2001, 276(44):41059-41063. |
| [6] | HOMMAIS A, RAYES J, HOULLIER A, et al. Molecular characterization of four ADAMTS13 mutations responsible for congenital thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome)[J]. Thromb Haemost, 2007, 98(3):593-599. |
| [7] | DE WAELE L, VERMEERSCH L, NGUYEN T T, et al. In vitro characterization of a novel Arg102 mutation in the ADAMTS13 metalloprotease domain[J]. J Thromb Haemost, 2023, 21(3):682-690. |
| [8] | JIANG Y, HUANG D, KONDO Y, et al. Novel mutations in ADAMTS13 CUB domains cause abnormal pre-mRNA splicing and defective secretion of ADAMTS13[J]. J Cell Mol Med, 2020, 24(7):4356-4361. |
| [9] | 丁秋兰, 王学锋. 遗传性易栓症的表型和基因诊断流程[J]. 诊断学理论与实践, 2019, 18(2):127-132. |
| DING Q L, WANG X L. The phenotype and flowchart of gene diagnosis in inherited thrombophilia[J]. J Diagn Concepts Pract, 2019, 18(2):127-132. | |
| [10] | 李蕾, 吴希, 戴菁, 等. 中国118例颅内静脉窦血栓患者的临床特点及危险因素分析[J]. 诊断学理论与实践, 2023, 22(3):261-269. |
| LI L, WU X, DAI J, et al. Clinical characteristics and risk factor analysis of 118 patients with cerebral venous sinus thrombosis[J]. J Diagn Concepts Pract, 2023, 22(3):261-269. | |
| [11] | 李蕾, 吴希, 许冠群, 等. 基于新一代测序技术的易栓症基因检测Panel的建立及其在中国静脉血栓患者遗传背景研究中的临床应用[J]. 诊断学理论与实践, 2019, 18(4):394-401. |
| LI L, WU X, XU G Q, et al. Establishment and applica tion of thrombophilia gene detection Panel based on next generation sequencing in identification of genetic back ground of Chinese patients with venous thromboembolism[J]. J Diagn Concepts Pract, 2019, 18(4):394-401. | |
| [12] | LIANG Q, ZHANG Z, DING B, et al. A noncanonical spli-cing variant c.875-5 T > G in von Willebrand factor causes in-frame exon skipping and type 2A von Willebrand disease[J]. Thromb Res, 2024, 236:51-60. |
| [13] | 金佩佩, 梁茜, 戴菁, 等. 一例2N型遗传性血管性血友病家系的表型诊断和基因型分析[J]. 诊断学理论与实践, 2018, 17(2):151-154. |
| JIN P P, LIANG Q, DAI J, et al. Phenotype and genotype analysis of a Chinese pedigree with 2N type von Wille-brand disease[J]. J Diagn Concepts Pract, 2018, 17(2):151-154. | |
| [14] | LIANG Q, QIN H, DING Q L, et al. Molecular and clinical profile of VWD in a large cohort of Chinese population: application of next generation sequencing and CNVplex? technique[J]. Thromb Haemost, 2017, 117(8):1534-1548. |
| [15] | BUDDE U, SCHNEPPENHEIM R, EIKENBOOM J, et al. Detailed von Willebrand factor multimer analysis in patients with von Willebrand disease in the European study, molecular and clinical markers for the diagnosis and management of type 1 von Willebrand disease (MCMDM-1VWD)[J]. J Thromb Haemost, 2008, 6(5):762-771. |
| [16] | EDVARDSEN M S, HANSEN E-S, UELAND T, et al. Impact of the von Willebrand factor-ADAMTS-13 axis on the risk of future venous thromboembolism[J]. J Thromb Haemost, 2023, 21(5):1227-1237. |
| [17] | TAYLOR A, VENDRAMIN C, SINGH D, et al. von Wille-brand factor/ADAMTS13 ratio at presentation of acute ischemic brain injury is predictive of outcome[J]. Blood Adv, 2020, 4(2):398-407. |
| [18] | LANCELLOTTI S, SACCO M, TARDUGNO M, et al. The von Willebrand factor-ADAMTS-13 axis:a two-faced Janus in bleeding and thrombosis[J/OL]. 2022[2025-01-20]. https://www.btvb.org/btvb/article/view/11. |
| [19] | PHILIPPE A, GENDRON N, BORY O, et al. Von Wille-brand factor collagen-binding capacity predicts in-hospital mortality in COVID-19 patients: insight from vWF/ADAMTS13 ratio imbalance[J]. Angiogenesis, 2021, 24(3):407-411. |
| [20] | AI J H, SMITH P, WANG S W, et al. The proximal carboxyl-terminal domains of ADAMTS13 determine substrate specificity and are all required for cleavage of von Willebrand factor[J]. J Biol Chem, 2005, 280(33):29428-29434. |
| [21] | FUJIMURA Y, MATSUMOTO M, KOKAME K, et al. Pregnancy-induced thrombocytopenia and TTP, and the risk of fetal death, in Upshaw-Schulman syndrome: a series of 15 pregnancies in 9 genotyped patients[J]. Br J Haematol, 2009, 144(5):742-754. |
| [22] | AKIYAMA M, TAKEDA S, KOKAME K, et al. Crystal structures of the noncatalytic domains of ADAMTS13 reveal multiple discontinuous exosites for von Willebrand factor[J]. Proc Natl Acad Sci U S A, 2009, 106(46):19274-19279. |
| [23] | HASSENPFLUG W A, OBSER T, BODE J, et al. Genetic and functional characterization of ADAMTS13 variants in a patient cohort with upshaw-schulman syndrome investigated in Germany[J]. Thromb Haemost, 2018, 118(4): 709-722. |
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