CN103820555A - 一种利用连锁不平衡在家系中筛查Wilson disease基因突变的方法 - Google Patents

一种利用连锁不平衡在家系中筛查Wilson disease基因突变的方法 Download PDF

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CN103820555A
CN103820555A CN201410070111.XA CN201410070111A CN103820555A CN 103820555 A CN103820555 A CN 103820555A CN 201410070111 A CN201410070111 A CN 201410070111A CN 103820555 A CN103820555 A CN 103820555A
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李卫东
杨付花
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Abstract

本发明公开了一种利用连锁不平衡在家系中筛查Wilson disease基因突变的方法，它基于连锁不平衡原理，以单核苷酸多态性基因分型的方法，依靠6个单核苷酸多态性位点确定肝豆状核变性患者以及其兄弟姐妹(或者三级以内亲属)的基因型状态，将家系其他成员的单倍型与先证者的单倍型相比较判断是否为患病或杂合携带者。

Description

一种利用连锁不平衡在家系中筛查Wilson disease基因突变的方法
技术领域
本发明属于分子生物学领域，具体而言，本发明涉及一种利用连锁不平衡在家系中筛查Wilson disease基因突变的方法，尤其是用于家系筛查的检测方法。 
背景技术
肝豆状核变性，又称Wilson Disease(WD)，是一种较为常见的常染色体隐性遗传病，其致病基因为ATP7B，在中国汉族人群的携带率为1/50.Wilson Disease的发病呈渐进性，如果能早期发现纯合性突变者，可以进行排铜治疗，但如果发现较晚，则会造成不可逆的神经损害.ATP7B基因较大，用DNA测序方法检测成本很高.临床多采用血浆铜蓝蛋白检测筛检，但其敏感性不及DNA测序，且携带者的检出是铜蓝蛋白检测的最大局限.曾有利用微卫星序列多态性检测ATP7B基因区域的单倍型来推测其突变情况，但微卫星序列的基因分型对设备的要求和成本均较高，难以用来进行人群水平的大规模筛检。 
目前，Wilson disease的诊断主要依赖于：临床表现包括神经和精神症状、旰异常、角膜色素环；辅助检查包括血浆铜蓝蛋白水平的降低、24小时尿铜排泄量增加、肝铜含量的增加以及核磁共振等影像学检查。但是这些临床表现常存在缺失，比如在幼儿患者中神经和精神症状在患病早期并不出现，并且生化指标的准确性也会受到其他因素的影响，比如外源性的雌激素或者怀孕期都可以使血清铜蓝蛋白的水平升高，这些因素也可以使Wilson disease患者个体的 血清铜蓝蛋白含量高于正常低值。现有的检查，没有一项能够作为确诊的指标，易出现误诊和漏诊。 
发明内容
为了克服临床表现的缺失和检查指标的不准确，本发明提供一种单倍型检测Wilson disease的方法，利用连锁不平衡在家系中快速筛查Wilson disease基因突变，该检测方法用于进行家系筛查，能够及早发现杂合携带者，也能够区分出无症状的患者和杂合携带者。 
本发明解决其技术问题所采用的技术方案是：选取与ATP7B(ATPase，Cu++transporting，beta polypeptide)基因突变呈连锁不平衡的6个单核苷酸多态性位点(Single Nucleotide Polymorphisms，SNP)包括rs1061472、rs2147363、rs1924611、rs7993492、rs1801243、rs28942074，以SNP基因分型的方法，不经过测序，仅依靠6个多态性位点确定Wilson disease患者个体兄弟姐妹(或三级以内的亲属)的基因突变状态。达到对先证者的家系的快速筛查，找出杂合携带者同时也区分出无症状者的目的。 
本发明的有益效果是，不经过测序，依靠6个多态性位点确定先证者家系中杂合携带者的同时，也能够区分出无症状者。可一次检测多个样本，成本低、快速。 
具体实施方式
在有患者的家系中，同时检测先证者以及兄弟姐妹(或三级以内亲属)的 基因突变状态。针对6个SNP位点设计Taqman探针，应用实时荧光定量聚合酶链反应(ploymerase chain reaction)鉴定每个位点的基因型状态。用以上6个SNP位点构建单倍型。比较先证者亲属与先证者的单倍型，Wilson Disease是常染色体隐性遗传病，若与先证者单倍型完全相同的为病人(纯合子)，完全不同的为正常个体，一半相同的为携带者(杂合子)。 
Figure BSA0000101481830000031
注：表格是6个SNP位点在染色体上的位置以及基因频率。 
本发明不需要经过DNA测序，依靠6个SNP位点来确定先证者以及家系成员的基因突变状态，可以同时检测数百例样本，所以用于家系的筛查简便快速。由于6个SNP位点存在连锁不平衡，物理距离小于0.1Mb，发生重组的可能性小于0.1％，具有较好的特异性。 
另外根据6个位点的基因频率可以计算出每个位点均为纯合的概率大约为0.002，这样6个位点检出的敏感度为99.8％ 
所以本发明的优点： 
1.简便快速，适合大样本筛检，成本较低； 
2.敏感度和特异性均较好； 
3.由于基因型在受精卵时期基因即已经确定，可用于新生儿的筛检，而铜蓝 蛋白的降低有一个逐渐发展的过程，且对突变携带者的检出不敏感，本发明有利于早期发现和早期排铜治疗。 
上述实施方式旨在举例说明本发明可为本领域专业技术人员实现或使用，对上述实施方式进行修改对本领域的专业技术人员来说将是显而易见的，故本发明包括但不限于上述实施方式，任何符合本权利要求书或说明书描述，符合与本文所公开的原理和新颖性、创造性特点的方法、工艺、产品，均落入本发明的保护范围之内。 

Claims (5)

1.一种利用连锁不平衡在家系中筛查Wilson disease基因突变的方法，其特征是：利用6个单核苷酸多态性位点的基因分型在家系中快速筛查Wilsondisease基因突变。
2.根据权利要求1所述的方法，其特征是：6个单核苷酸多态性位点与ATP7B基因的突变呈连锁不平衡。
3.根据权利要求1所述的方法，其特征是：6个单核苷酸多态性位点包括rs1061472、rs2147363、rs1924611、rs7993492、rs1801243、rs28942074。
4.根据权利要求1-3所述的方法，其特征在于Taqman探针，应用实时荧光定量聚合酶链反应(ploymerase chain reaction)鉴定每个位点的基因型状态；用以上6个SNP位点构建单倍型。比较先证者亲属与先证者的单倍型。
5.根据权利要求4所述的方法，其特征在于所述的方法是非诊断目的的。
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CN109055533A (zh) * 2018-09-12 2018-12-21 中国人民解放军陆军军医大学第附属医院 一种检测atp7b基因突变的引物组合、检测方法及试剂盒
CN116004668A (zh) * 2022-08-01 2023-04-25 湖南家辉生物技术有限公司 一种ATP7B基因致病突变体及其在制备Wilson病诊断试剂盒中的应用

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108220403A (zh) * 2017-12-26 2018-06-29 北京科迅生物技术有限公司 特定突变位点的检测方法、检测装置、存储介质及处理器
CN108220403B (zh) * 2017-12-26 2021-07-06 北京科迅生物技术有限公司 特定突变位点的检测方法、检测装置、存储介质及处理器
CN109055533A (zh) * 2018-09-12 2018-12-21 中国人民解放军陆军军医大学第附属医院 一种检测atp7b基因突变的引物组合、检测方法及试剂盒
CN116004668A (zh) * 2022-08-01 2023-04-25 湖南家辉生物技术有限公司 一种ATP7B基因致病突变体及其在制备Wilson病诊断试剂盒中的应用
CN116004668B (zh) * 2022-08-01 2024-05-31 湖南家辉生物技术有限公司 一种ATP7B基因致病突变体及其在制备Wilson病诊断试剂盒中的应用

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