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

Establishment and application of thrombophilia gene detection panel based on next generation sequencing in identification of genetic background of Chinese patients with venous thromboembolism

目的:研发髙效、准确、简单、实用的易栓症遗传性危险因素的基因检测方法 ,用于我国易栓症患者的病因诊断和治疗指导。方法:收集上海交通大学医学院附属瑞金医院血栓与止血门诊246例静脉血栓患者的临床及家系资料,并对其进行遗传性及获得性易栓症表型检测;通过文献研究和人类孟德尔遗传学数据库检索,遴选出18种与血栓发生相关的基因,组成易栓症基因检测Panel,采用二代测序及CNVplex高通量拷贝数检测技术对该Panel进行点突变、小缺失/插入及拷贝数变异检测。结果:246例静脉血栓患者中有159例携带基因突变,突变检出率为64.6%。其中,69.2%的患者携带单一基因突变,13.2%携带单基因的复合杂合突变,另有17.6%携带2种及以上的基因突变。在159例携带基因突变的患者中,有144例患者携带抗凝蛋白基因(SERPINC1、PROS1和PROC)的突变,有31例患者携带其他10种基因(F2、F5、F9、F12、PROCR、THBD、SERPIND1、PLG、ADAMTS13和TFPI)的突变,其中部分突变(F2基因R596Q和F9基因R384Q突变等)被证实与静脉血栓发生相关。拷贝数检测发现,有19例患者存在拷贝数变异,以PROCR及PROS1基因为主。另外,在61例存在获得性血栓危险因素(抗磷脂综合征、手术或妊娠等)的患者中,有40例患者携带遗传性血栓危险因素。56例患者实验室表型检测结果均为正常,而易栓症基因检测Panel结果显示,其中20例患者携带致病性基因突变。72例患者因处于血栓急性期或口服抗凝药物期间,无法进行表型检测,经易栓症基因检测Panel分析发现,其中32例患者携带致病性基因突变。结论:本研究建立的易栓症基因检测Panel可以快速、有效、准确地对遗传性静脉血栓危险因素进行筛查。对存在获得性血栓危险因素的患者仍有必要进行遗传分析。根据基因检测结果,可评估患者及其家系成员血栓发生风险的大小,制定相应的预防治疗方案,预防血栓的发生或复发,减少血栓后综合征的发生,值得在临床上广泛推广应用。

Objective: To develop an accurate, simple and practical method for detecting hereditary risk factors of thrombophilia, which can be used for early diagnosis and treatment guidance in Chinese thrombophilia patients. Methods:Altogether 246 patients with venous thrombosis at our hospital were enrolled. The clinical data and family history were collected. Phenotypic examinations were performed. Eighteen candidate genes related to thrombosis were selected to compose a gene panel through literature searching. Point mutation, small deletion/insertion and copy number variations of this panel were detected by next-generation sequencing and CNVplex technique. Results: Of the 246 patients with venous thrombosis, 159 patients were identified as having gene mutations. The mutation detection rate was 64.6%. Among them, 69.2% carried a single mutation, 13.2% carried compound heterozygous mutation in one gene, and 17.6% carried mutations of at least two genes. Of the 159 patients carrying gene mutations,144 patients carried mutations of anticoagulant protein genes(SERPINC1, PROS1 and PROC), while 31 patients carried mutations of other 10 genes(F2, F5, F9, F12,PROCR, THBD, SERPIND1, PLG, ADAMTS13 and TFPI), in which some mutations(F2 R384 Q and F9 R596 Q) had been confirmed to be associated with venous thrombosis. Copy number detection showed that 19 patients had copy number variations, mainly in PROCR and PROS1 genes. In addition, 40 of 61 patients with acquired thrombosis risk factors(antiphospholipid syndrome, surgery or pregnancy, etc.) carried hereditary thrombosis risk factors. Of the 56 patients with normal phenotypic results, 20 of them carried pathogenic thrombotic mutations revealed by genetic analysis. Moreover, of the 72 patients who were in the acute stage of thrombosis or during anticoagulants treatment thus could not take phenotypic examination, 32 were identified as having pathogenic thrombotic mutations. Conclusions: The hemophilia gene detection panel established can screen the hereditary thrombosis risk factors more quickly, effectively and accurately. It is necessary to carry out genetic analysis in patients with acquired risk factors. According to the results of gene analysis,clinicians can provide appropriate preventive treatment to prevent the occurrence or recurrence of thrombosis, and to reduce the occurrence of post-thrombotic syndrome, and is worthy of application in clinical practice.