两个遗传性抗凝血酶缺陷症家系的表型与基因突变分析  

Phenotypic and genetic analysis of two pedigrees affected with hereditary antithrombin deficiency

作　　者：郝秀萍[1] 金艳慧[1] 程晓丽[1] 杨丽红[1] 朱丽青[1] 王明山[1] 

机构地区：[1]浙江省温州医科大学附属第一医院医学检验中心,325000

出　　处：《中华医学遗传学杂志》2016年第2期145-149,共5页Chinese Journal of Medical Genetics

基　　金：温州市科技计划项目（Y20150302）

摘　　要：目的对两个遗传性抗凝血酶（antithrombin，AT）缺陷症家系进行表型诊断和基因分析，探讨其分子发病机制。方法检测2例先证者及其家系成员凝血酶原时间、活化部分凝血活酶时间、凝血酶时间、纤维蛋白原、抗凝血酶活性（antithrombinactivity，AT：A）、抗凝血酶抗原（antithrombinantigen，AT：Ag）、蛋白C活性及蛋白S活性等指标以明确诊断。提取外周血基因组DNA，PCR扩增AT基因全部外显子、侧翼及5′、3′非编码区，PCR产物经纯化后直接测序，寻找基因异常位点，并通过反向测序及银染色进行验证。借助生物信息学软件辅助分析突变对蛋白的影响。结果先证者1的AT：Ag正常但AT：A降低至30％，其大儿子、小儿子、小女儿AT：A在正常值的50％左右;AT基因第2外显子和第5外显子分别存在杂合错义突变c．235C〉T（P．Arg47Cys）和两个多态性位点（c．981G〉A、c．1011G〉A），同样突变也见于其大儿子、小儿子和小女儿。先证者2的AT：A、AT：Ag分别为39％和103mg／L，其父亲为57％和114mg／L，均明显低于正常对照；AT基因第3外显子发生杂合缺失突变g．5890—5892delctt，导致P．Phe121丢失，其父亲亦携带该突变位点。SIFT和PolyPhen-2程序对C．235C〉T分析表明，错义突变Arg47Cys会对AT蛋白功能产生影响；spdbv软件和PIC程序对g．5890—5892delCTT分析显示，缺失突变导致Phe121与Ala124、Lys125的氢键连接以及与Lys125、Arg47的阳离子-π作用力消失，从而影响蛋白稳定性。结论先证者1表现为Ⅱ型AT缺陷，先证者2表现为Ⅰ型AT缺陷；两例先证者抗凝血酶水平可能与错义突变P．Arg47Cys及缺失突变g．5890-5892delCTT有关。Objective To explore the phenotype, genotype and molecular mechanism for two pedigrees affected with hereditary antithrombin （AT） deficiency. Methods- Clinical diagnosis was＇validated by assaying of coagulation parameters including prothrombin time, activated parfial thromboplastin time, thromhin time, fibrinogen, antithrombin activity （AT： A） and specific antigen（AT：Ag）, protein C activity, as well as protein S activity. To detect potential mutations in the probands, all exons, exon-intron boundaries and the 3′,5′untranslated regions were amplified by PCR and subjected to direct sequencing. Suspected mutation was confirmed by reverse sequencing and silver staining. The effect of mutations on the AT protein was analyzed with bioinformatics software. Results The AT：Ag of pedigree 1 was normal, but its AT： A has reduced to 30%. A heterozygous c. 235C〉T mutation in exon 2 causing p. Arg47Cys, in addition with two single nucleotide polymorphisms （e. 981G〉A, c. 1011G〉A） in exon 5 were identified in the patient. His four children, except for the elder daughter, were heterozygous for the mutations. The plasma levels of AT：A and AT：Ag in proband 2 have decreased to 39% and 103 mg/L, respectively. A heterozygous deletion （g. 5890-5892delCTT） leading to loss of p. Phe121 was also detected in his father. Bioinformatie analysis suggested that the missense mutation Arg47Cys can affect the functions of AT protein. Meanwhile, lacking of Phe121 will result in loss of hydrogen bonds with Ala124, Lys125 and thecation-π interactions with Lys125, Arg47, which may jepordize the stability of the protein. Conclusion The proband 1 had type Ⅱ AT deficiency, while proband 2 had type Ⅰ AT deficiency. The p. Arg47Cys and g. 5890-5892delCTT mutations of the AT gene are significantly correlated with the levels of AT in the two probands, respectively.

关 键 词：抗凝血酶缺陷症 基因突变 血栓形成 

分 类 号：R554[医药卫生—血液循环系统疾病] R394[医药卫生—内科学]