9例遗传性凝血因子Ⅶ缺陷症患者的基因诊断与表型分析

doi:10.3969/j.issn.1673-8640.2016.04.007

摘要/Abstract

摘要：

目的探讨9例遗传性凝血因子Ⅶ （FⅦ）缺陷症患者的基因突变类型与临床特征。方法采用一期法检测患者的FⅦ活性（FⅦ：C）,用双抗体夹心酶联免疫吸附试验（ELISA）检测其FⅦ抗原（FⅦ：Ag）水平,并抽提患者的外周血基因组DNA,聚合酶链反应（PCR）扩增FⅦ基因所有外显子及其侧翼序列,采用DNA直接测序进行基因分析。结果在9例遗传性FⅦ 缺陷症患者中发现10种基因突变,包括3种剪切位点突变和7种错义突变。1例患者为p.Tyr128（68）Cys纯合突变,其FⅦ:C为0.8%,FⅦ：Ag为2.5%,临床表型为反复重度出血;5例患者携带FⅦ基因双杂合突变,分别为p.Thr241（181）Asn和p.Gly406（346）Asn、IVS1a+5G>A和p.His408（348）Gln、IVS5-1G>A和p.His408（348）Gln、c.*64G>A合并p.Ile213（153）Asn、p.Cys389（329）Gly和p.His408（348）Gln的双杂合突变,其相应的FⅦ：C分别为1.2%、4.4%、1.0%、0.5%和1.2%,患者临床出血症状轻重不一;3例患者携带杂合突变,分别为p.Cys389（329）Gly、p.His408（348）Gln和p.Thr419（359）Met,其FⅦ：C分别为0.5%、8.3%和9.4%,第1位有出血史,后2位无明显出血。结论在9例遗传性FⅦ缺陷症患者中发现了10种基因突变,其中p.Gly406（346）Asn、c.*64G>A、p.Ile213（153）Asn为新发现突变。p.His408（348）Gln突变较常见,而FⅦ:C与患者的临床表型间无相关性。

关键词: 遗传性凝血因子Ⅶ缺陷症, 基因突变, 表型

Abstract:

Objective To investigate the gene mutations of coagulation factor Ⅶ（FⅦ） and the clinical characteristics in 9 patients with hereditary FⅦ deficiency. Methods FⅦ activity（FⅦ：C） and FⅦ antigen （FⅦ：Ag） were determined by one-stage clotting test and double-antibody sandwich enzyme-linked immunosorbent assay （ELISA）,respectively. Genomic DNA was extracted from peripheral blood. All the exons and flank sequences of FⅦ gene were amplified by polymerase chain reaction （PCR）,and gene analysis was performed by direct sequencing. Results A total of 10 different mutations were identified in 9 patients with hereditary FⅦ deficiency,including 3 splice site mutations and 7 miss sense mutations. One patient had p.Tyr128（68）Cys homozygous mutation,FⅦ：C was 0.8%,FⅦ：Ag was 2.5%,and the clinical characteristic was severe bleeding. Five patients had double heterozygous mutations,p.Thr241（181）Asn with p.Gly406（346）Asn,IVS1a+5G>A with p.His408（348）Gln,IVS5-1G>A with p.His408（348）Gln,c.*64G>A with p.Ile213（153）Asn and p.Cys389（329）Gly with p.His408（348）Gln,and FⅦ：C were 1.2%,4.4%,1.0%,0.5% and 1.2%,respectively. The clinical bleeding symptoms had various severities. Three patients had mono-heterozygous mutations,p.Cys389（329）Gly,p.His408（348）Gln and p.Thr419（359）Met,and FⅦ：C were 0.5%,8.3% and 9.4%,respectively. The first patient had a history of bleeding,and the other 2 patients had no significant bleeding. Conclusions A total of 10 types of gene mutations are identified in 9 patients with hereditary FⅦ deficiency. p.Gly406（346）Asn,c.*64G>A and p.Ile213（153）Asn are found newly,and p.His408（348）Gln is a common mutation,and FⅦ：C has no correlation with clinical phenotypes.

Key words: Hereditary coagulation factor Ⅶ deficiency, Gene mutation, Phenotype

中图分类号:

R446.11

陆一一, 丁秋兰, 戴菁, 王剑飚, 蔡晓红, 王学锋. 9例遗传性凝血因子Ⅶ缺陷症患者的基因诊断与表型分析[J]. 检验医学, 2016, 31(4): 275-281.

LU Yiyi, DING Qiulan, DAI Jing, WANG Jianbiao, CAI Xiaohong, WANG Xuefeng. Genetic diagnosis and phenotype analysis for 9 patients with hereditary coagulation factor Ⅶ deficiency[J]. Laboratory Medicine, 2016, 31(4): 275-281.

图/表 4

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外显子	引物序列	引物位置	扩增片段长度（bp）
1a	F：GCTCAGTGGCTGGGCAGCAG R：CAAGGGCGGGGTTTGGGAAG	g.4863-4882 g.5186-5205	311
1b	F：AGTGGGGGGTGGGCTGTGAGG R：CACCCTCCTTGGGGGGCACTG	g.5958-5978 g.6223-6243	178
2	F：GGCGGTCTCCGAGGCACTGG R：GTGAACCAGGCCGCGTGGG	g.9837-9856 g.10175-10193	291
3+4	F：AACCCCAGTTCATGGTGTGTC R：ATCAGAACCTTGGGTCCCTGT	g.12875-12895 g.13353-13373	540
5	F：TCAGACATGTGGCTCCTCTGA R：CCACCCGTCTTTTGTCCAGTA	g.14665-14685 g.15073-15093	271
6	F：CCCTGACCATTGTCTCCTCAG R：CCTTCCTTGCCCCACTAAAAG	g.15733-15753 g.16244-16264	259
7	F：AAATTGTGCAATGCCAGAGGT R：CTCAAGTGTCTCAGGGGCTGT	g.16580-16600 g.17102-17122	482
8	F：AGCCCATCCCCATGCACCAG R：CCTCCCTCCCATGCCCTCCT	g.17550-17569 g.18312-18331	876
5'非翻译区	F：AGGCTCTCTTCAAATATTTACATC R：CGGGCTGGCTCCTGGATTT	g.4612-4635 g.5165-5183	571
3'非翻译区	F：AAGCTCATGCGCTCAGAGCC R：CCTGGGAGCTACAGCCTCAA	g.18162-18181 g.19383-19402	1 296

外显子	引物序列	引物位置	扩增片段长度（bp）
1a	F：GCTCAGTGGCTGGGCAGCAG R：CAAGGGCGGGGTTTGGGAAG	g.4863-4882 g.5186-5205	311
1b	F：AGTGGGGGGTGGGCTGTGAGG R：CACCCTCCTTGGGGGGCACTG	g.5958-5978 g.6223-6243	178
2	F：GGCGGTCTCCGAGGCACTGG R：GTGAACCAGGCCGCGTGGG	g.9837-9856 g.10175-10193	291
3+4	F：AACCCCAGTTCATGGTGTGTC R：ATCAGAACCTTGGGTCCCTGT	g.12875-12895 g.13353-13373	540
5	F：TCAGACATGTGGCTCCTCTGA R：CCACCCGTCTTTTGTCCAGTA	g.14665-14685 g.15073-15093	271
6	F：CCCTGACCATTGTCTCCTCAG R：CCTTCCTTGCCCCACTAAAAG	g.15733-15753 g.16244-16264	259
7	F：AAATTGTGCAATGCCAGAGGT R：CTCAAGTGTCTCAGGGGCTGT	g.16580-16600 g.17102-17122	482
8	F：AGCCCATCCCCATGCACCAG R：CCTCCCTCCCATGCCCTCCT	g.17550-17569 g.18312-18331	876
5'非翻译区	F：AGGCTCTCTTCAAATATTTACATC R：CGGGCTGGCTCCTGGATTT	g.4612-4635 g.5165-5183	571
3'非翻译区	F：AAGCTCATGCGCTCAGAGCC R：CCTGGGAGCTACAGCCTCAA	g.18162-18181 g.19383-19402	1 296

患者	1	2	3	4	5	6	7	8	9
PT（s）	38.6	54.0	23.8	31.5	59.7	34.0	28.4	21.6	36.1
FⅦ：C（%）	1.2	0.5	4.4	1.0	0.8	0.5	1.2	8.3	9.4
FⅦ：Ag（%）		50.0	2.0	5.0	2.5		20.0	30.0

患者	1	2	3	4	5	6	7	8	9
PT（s）	38.6	54.0	23.8	31.5	59.7	34.0	28.4	21.6	36.1
FⅦ：C（%）	1.2	0.5	4.4	1.0	0.8	0.5	1.2	8.3	9.4
FⅦ：Ag（%）		50.0	2.0	5.0	2.5		20.0	30.0

患者	外显子/内含子	核苷酸突变	氨基酸变化	首次报道（年份）	突变类型
1	7 8	g.16723C>A g.18034G>A	p.Thr241（181）Asn p.Gly406（346）Asn	Millar（2000）本研究	双杂合
2	8	g.17982T>G	p.Cys389（329）Gly	Wu（2000）	杂合
3	IVS1a 8	c.64+5G>A g.18041T>G	p.His408（348）Gln	Peyvandi（2000） Katsumi（2000）	双杂合
4	IVS5 8	c.572-1G>A g.18041T>G	p.His408（348）Gln	Yu（2009） Katsumi（2000）	双杂合
5	4	g.13123A>G	p.Tyr128（68）Cys	Karin（2000）	纯合
6	IVS1a 6	c.*64G>A g.16042T>A	p.Ile213（153）Asn	本研究本研究	双杂合
7	8 8	g.17982T>G g.18041T>G	p.Cys389（329）Gly p.His408（348）Gln	Wu（2000） Katsumi（2000）	双杂合
8	8	g.18041T>G	p.His408（348）Gln	Katsumi（2000）	杂合
9	8	g.18073C>T	p.Thr419（359）Met	Arbini（1996）	杂合