6个遗传性凝血因子Ⅹ缺陷症家系的表型与基因型诊断

doi:10.16150/j.1671-2870.2020.01.006

摘要/Abstract

摘要：

目的：探讨6个遗传性凝血因子Ⅹ（factor X, FⅩ）缺陷症家系的分子发病机制,并采用凝血酶生成试验（thrombin generation test, TGT）评估FⅩ缺陷症患者的出血风险。方法：收集6例先证者及相关家系成员的临床资料,凝固法检测内源性、外源性以及共同途径激活的FⅩ活性（FⅩ∶C）,酶联免疫吸附试验双抗体夹心法检测FⅩ抗原（FⅩ∶Ag）,采用蛋白印迹法检测血浆中的FⅩ相对分子质量及含量,直接测序法检测FⅩ基因（F10）突变,采用TGT检测6例先证者及部分家系成员血浆中的凝血酶生成。结果：在6例先证者中共发现了10种F10突变,其中5种（p.Cys246Ser, p.Tyr319Cys, p.Leu252Phe, p.Arg313Gln以及IVS5-2A>G）为新突变,其余5种（p.Phe71Ser, p.Val338Met, p.Gly406Ser, p.Gly365Ser, p.Cys151Arg）为已报道突变。TGT显示凝血酶生成潜力（endogenous thrombin potential, ETP）及峰值这2个参数与FⅩ缺陷症患者临床出血表现的严重程度具有一定关联。结论：共发现5种F10基因新突变;5例遗传性FⅩ缺陷症患者由F10双杂合突变所致,1例由F10单杂合突变所致;在浓度为1 pM （1 pmol/L）组织因子（tissue factor,TF）激活下的TGT中,测得的ETP及峰值这2个参数可用于评估FⅩ缺陷症患者的出血倾向,对于了解该病患者的临床出血风险有一定的参考价值。

关键词: 凝血因子Ⅹ缺陷症, 凝血酶生成试验, 表型, 基因型

Abstract:

Objective: To investigate the molecular pathogenesis of 6 pedigrees with inherited coagulation factorⅩ (FⅩ) deficiency and to assess the bleeding risk in patients with inherited FⅩ deficiency by thrombin generation test (TGT).Methods: Clinical data of the 6 pedigrees were collected. The FⅩ coagulation activity (FⅩ:C) was tested by clotting test and FⅩ antigen (FⅩ:Ag) was detected by double antibody sandwich enzyme-linked immunosorbent assay（ELISA）. The concentration of FⅩ in plasma was measured with Western blotting. The FⅩ gene (F10) mutation was analyzed by direct sequencing. Thrombin generation (TG) in six patients and some family members was measured using TET. Results: Ten F10 mutations were identified in 6 probands, five of which (p.Cys 246 Ser, p.Tyr 319 Cys, p.Leu 252 Phe, p.Arg 313 Gln and p. IVS5-2A>G) were novel and the other five(p.Phe71Ser, p.Val 338 Met, p.Gly 406 Ser, p.Gly 365 Ser and p.Cys 151 Arg) had been previously reported. TGT showed that endogenous thrombin potential (ETP) and peak height (Peak) had certain correlation with bleeding severity of FX-deficient patients. Conclusions: Five noval mutations of FⅩ are identified. Five patients were resulted from compound heterozygous F10 mutations, while the remaining one is caused by one heterozygous F10 mutation. TGT parameters including ETP and Peak could be used to assess the bleeding risk in patients with FⅩ deficiency, and TGT might serve as a useful laboratory tool to monitor the risk of bleeding.

Key words: Coagulation factor Ⅹ deficiency, Thrombin generation test, Phenotype, Genotype

中图分类号:

R446

刘佳婕, 邵妍妍, 梁茜, 丁秋兰. 6个遗传性凝血因子Ⅹ缺陷症家系的表型与基因型诊断[J]. 诊断学理论与实践, 2020, 19(1): 20-27.

LIU Jiajie, SHAO Yanyan, LIANG Qian, DING Qiulan. Phenotype and genotype diagnosis of 6 pedigrees with inherited coagulation factor Ⅹ deficiency[J]. Journal of Diagnostics Concepts & Practice, 2020, 19(1): 20-27.

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参考文献 20

[1]	Hertzberg M. Biochemistry of factor Ⅹ[J]. Blood Rev, 1994, 8(1):56-62. pmid: 8205010
[2]	Jackson CM. Factor Ⅹ[J]. Prog Hemost Thromb, 1984, 7:55-109. pmid: 6397768
[3]	Uprichard J, Perry DJ. Factor Ⅹ deficiency[J]. Blood Rev, 2002, 16(2):97-110. pmid: 12127953
[4]	Girolami A, Cosi E, Santarossa C, et al. Factor Ⅹ friuli coagulation disorder: almost 50 years later[J]. Clin Appl Thromb Hemost, 2018, 24(1):33-40. doi: 10.1177/1076029616686423 URL
[5]	Menegatti M, Peyvandi F. Factor Ⅹ deficiency[J]. Semin Thromb Hemost, 2009, 35(4):407-415. doi: 10.1055/s-0029-1225763 pmid: 19598069
[6]	Dorgalaleh A, Zaker F, Tabibian S, et al. Spectrum of factor Ⅹ gene mutations in Iranian patients with congenital factor Ⅹ deficiency[J]. Blood Coagul Fibrinolysis, 2016, 27(3):324-327. doi: 10.1097/MBC.0000000000000435 URL
[7]	Brown DL, Kouides PA. Diagnosis and treatment of inherited factor Ⅹ deficiency[J]. Haemophilia, 2008, 14(6):1176-1182. doi: 10.1111/j.1365-2516.2008.01856.x pmid: 19141158
[8]	Peyvandi F, Menegatti M, Santagostino E, et al. Gene mutations and three-dimensional structural analysis in 13 families with severe factor Ⅹ deficiency[J]. Br J Haematol, 2002, 117(3):685-692. doi: 10.1046/j.1365-2141.2002.03486.x URL
[9]	Girolami A, Vettore S, Scarparo P, et al. Persistent vali-dity of a classification of congenital factor Ⅹ defects based on clotting, chromogenic and immunological assays even in the molecular biology era[J]. Haemophilia, 2011, 17(1):17-20. doi: 10.1111/j.1365-2516.2010.02328.x pmid: 20546029
[10]	Duarte RCF, Rios DRA, Rezende SM, et al. Standardization and evaluation of the performance of the thrombin generation test under hypo- and hypercoagulability conditions[J]. Hematol Transfus Cell Ther, 2019, 41(3):244-252.
[11]	Tripodi A. Thrombin generation assay and its application in the clinical laboratory[J]. Clin Chem, 2016, 62(5):699-707. doi: 10.1373/clinchem.2015.248625 URL
[12]	Joly BS, Sudrié-Arnaud B, Barbay V, et al. Thrombin generation test as a marker for high risk venous thrombosis pregnancies[J]. J Thromb Thrombolysis, 2018, 45(1):114-121. doi: 10.1007/s11239-017-1572-3 URL
[13]	Liang Q, Chen Q, Ding Q, et al. Six novel missense mutations causing factor Ⅹ deficiency and application of thrombin generation test[J]. Thromb Res, 2013, 131(6):554-9. doi: 10.1016/j.thromres.2013.04.014 pmid: 23664564
[14]	The F10 gene mutation database[R/OL]. [2020-01-31]. http://www.hgmd.cf.ac.uk/ac/all.php.
[15]	Millar DS, Elliston L, Deex P, et al. Molecular analysis of the genotype-phenotype relationship in factor Ⅹ deficiency[J]. Hum Genet, 2000, 106(2):249-257. pmid: 10746568
[16]	Borhany M, Buthiau D, Rousseau F, et al. Genotyping of five Pakistani patients with severe inherited factor Ⅹ deficiency: identification of two novel mutations[J]. Blood Coagul Fibrinolysis, 2018, 29(7):622-625. doi: 10.1097/MBC.0000000000000764 URL
[17]	Isshiki I, Favier R, Moriki T, et al. Genetic analysis of hereditary factor Ⅹ deficiency in a French patient of Sri Lankan ancestry: in vitro expression study identified Gly366Ser substitution as the molecular basis of the dysfunctional factor Ⅹ[J]. Blood Coagul Fibrinolysis, 2005, 16(1):9-16. doi: 10.1097/00001721-200501000-00002 URL
[18]	Miyata T, Kojima T, Suzuki K, et al. Factor Ⅹ Nagoya 1 and Nagoya 2: a CRM- factor Ⅹ deficiency and a dysfunctional CRM+ factor Ⅹ deficiency characterized by substitution of Arg306 by Cys and of Gly366 by Ser, respectively[J]. Thromb Haemost, 1998, 79(3):486-490. doi: 10.1055/s-0037-1614930 URL
[19]	Mota L, Shetty S, Idicula-Thomas S, et al. Molecular basis of factor Ⅹ deficiency cases from India[J]. Haemophilia, 2010, 16(4):693-697. doi: 10.1111/j.1365-2516.2010.02213.x pmid: 20331754
[20]	Herrmann FH, Auerswald G, Ruiz-Saez A, et al. Factor Ⅹ deficiency: clinical manifestation of 102 subjects from Europe and Latin America with mutations in the factor 10 gene[J]. Haemophilia, 2006, 12(5):479-489. pmid: 16919077

先证者	性别/ 年龄（岁）	临床表现	出血评分(分)	APTT (s)	PT (s)	FⅩ∶C			FⅩ∶ Ag	分型	基因检测
先证者	性别/ 年龄（岁）	临床表现	出血评分(分)	APTT (s)	PT (s)	APTT途径	PT途径	RVV途径	FⅩ∶ Ag	分型	基因检测
1	男/10	皮肤淤斑,换牙时出血, 肌肉出血	7	74.1	28.9	2.4	2.6	2.4	7.3	Ⅰ型	c.212T>C, p.Phe71Ser; c.1012G>A,p.Val338Met
2	女/79	月经量多,手术后出血难止,拔牙后出血	4	42.7	15.8	38.9	42.2	44.4	83.1	Ⅱ型	c.1216G>A, p.Gly406Ser
3	女/36	月经量多,外伤后出血难止	6	48.3	20.2	15.8	16.8	12.3	67.6	Ⅱ型	c.736T>A, p.Cys246Ser; c.956A>G, p.Tyr319Cys
4	女/24	自发鼻出血,皮肤易发淤斑,牙龈出血	4	55.9	26.5	4.2	7.2	6.2	35.4	Ⅱ型	c.1093G>A, p.Gly365Arg; c.754C>T, p.Leu252Phe
5	男/13	无出血症状	0	38.8	16.3	27.9	12.7	16.7	18.2	Ⅰ型	c.938G>A, p.Arg313Gln; IVS5-2A>G
6	男/26	鼻出血,牙龈出血,皮肤易发淤斑,关节出血,肌肉血肿	10	52.5	30.6	3.8	2.4	2.5	5.6	Ⅰ型	c.212T>C, p.Phe71Ser; c.451T>C, p.Cys151Arg

患者及家系成员	LT(min)	ETP (nmol*min)	峰值(nmol)	TTP(min)
P1-(Ⅱ-1)	13.33	98.23	3.77	28.67
P1-(Ⅰ-1)	7.00	408.12	41.61	12.67
P1-(Ⅰ-2)	6.76	626.77	33.18	17.12
P2-(Ⅱ-1)	7.83	763.50	55.81	15.17
P3-(Ⅱ-1)	10.33	224.32	15.60	17.67
P3-(Ⅲ-1)	9.00	944.50	88.70	14.67
P4-(Ⅱ-1)	30.32	99.90	5.04	40.84
P5-(Ⅱ-1)	7.83	295.56	26.57	13.83
P5-(Ⅰ-1)	6.00	764.93	68.50	12.00
P6-(Ⅱ-1)	5.17	83.91	3.04	14.67
正常对照者	6.17	969.31	92.49	11.67