本发明涉及基因及其检测方法，特别涉及第220位点发生突变的人类ibgc致病基因pdgfb及其检测方法。
背景技术：
人类特发性基底节钙化(idiopathicbasalgangliacalcification,ibgc)是一种以影像学上基底节及脑其它部位对称性钙化为特征的神经系统变性遗传病,也称fahr病。钙化最常见的部位为基底节区豆状核，尤其是苍白球，也常见于壳核、丘脑、尾状核及小脑齿状核等部位，甚至出现在大脑皮层及皮层下白质。ibgc属罕见病，呈散发或家族性发病，家族性患者发病年龄逐代提前，进展缓慢、病程较长。该病无明显性别差异，表现为复杂多样的运动损害和行为症状的组合，同一家族中的不同成员之间临床症状往往存在差异。临床表现以神经、精神功能紊乱为主，轻者仅仅为轻度注意力或记忆力的下降，重者则会出现人格及行为改变，终致精神病或痴呆。ibgc有家族性发病的现象，常染色体显性遗传和隐性遗传的病例均有报道，以常染色体显性遗传居多。遗传性ibgc相关致病基因和发病机制长期以来并未得到解析，直到2012年liu等人在ibgc3基因位点中，通过连锁分析、单倍体分析、sanger测序等成功克隆特发性基底节钙化的致病基因slc20a2，并且在不同种族中得到验证；此后，pdgfrb、pdgfb和xpr1这三个致病基因陆续被发现和得到验证。其中，slc20a2基因编码的蛋白为钠-磷共转运体(pit2)，参与机体的钙磷调节；pdgfrb基因编码血小板衍生生长因子受体pdgfrβ，属于iii型受体酪氨酸激酶家族；pdgfb基因则编码血小板衍生生长因子pdgf-b，与pdgfbβ结合可激活下游信号通路，参与细胞的增殖、分化、生存以及迁移；xpr1基因编码的蛋白含有spx结构域，主要参与磷稳态的维持，具有pi输出功能。为了系统解析ibgc致病基因突变，本发明设计和合成了覆盖4个已知致病基因的pcr捕获引物组，并结合二代测序技术对10个ibgc患者样本进行捕获测序，共分析得出了1种slc20a2基因突变、2种pdgfrb基因突变、2种pdgfb基因突变和2种xpr1基因突变，所发现的突变均经sanger测序确证；之后针对各自的致病基因突变，利用sanger测序分析对应的家系中其它成员的样本，全部符合基因型-表型共分离规律从而致病基因突变得到了验证。进一步的，采用同样的方法在另外12个ibgc患者和200个正常人样本中进行了验证。本发明提供的ibgc致病基因突变尚未见报道，其对于该病的解析、筛查、检测和治疗具有潜在的重要价值。此外，本发明基于pcr捕获结合二代测序(简称pcr捕获测序)构建的ibgc致病基因检测方法，可以同时全面解析4种ibgc致病基因突变谱，具有通量高、灵敏度高、可解析未知突变的优点。目前，国内外均没有该方法在ibgc致病基因slc20a2，pdgfrb，pdgfb，xpr1突变同时检测方面应用的报道。由于单次反应测序长度的限制(1000bp以内)，传统的基于sanger测序的方法进行的全面解析，费用高昂且费时费力。而基于实时荧光定量pcr的突变检测方法，则只能针对少量已知的突变。技术实现要素：本发明提供了ibgc相关的4个致病基因的7种突变基因形式，同时提供了基于pcr捕获测序的检测方法；这些可以为ibgc致病机理的解析和药物开发、致病基因筛查和检测以及治疗方案的制定等提供依据和奠定基础。本发明解决其技术问题所采用的技术方案如下：本发明提供了ibgc的7种致病基因形式，包括1种slc20a2突变基因，其序列如seqidno.1所示；2种pdgfrb突变基因，其序列如seqidno.2和seqidno.3所示；2种pdgfb突变基因，其序列如seqidno.4和seqidno.5所示；和2种xpr1突变基因，其序列如seqidno.6和seqidno.7所示。所述涉及slc20a2、pdgfrb、pdgfb和xpr1这4个基因共7种突变基因类型经检测和验证，均为ibgc致病基因。其中，slc20a2突变基因的突变位点在exon7中，具体是野生型基因第799位的g突变为c；pdgfrb突变基因的突变位点其一在exon2中，具体是野生型基因第3位的g突变为a,其二在exon16中，具体是野生型基因第2209位的g突变为a；pdgfb突变基因的突变位点其一在exon3中，具体是野生型基因第220位的g突变为t，其二也在exon3中，具体是野生型基因第232位的c突变为t；xpr1突变基因的突变位点其一在exon5中，具体是野生型基因第490位的g突变为t，其二在exon13中，具体是野生型基因第1708位的c突变为t。由于突变发生在外显子区，因此本发明提供的seqidno.1-7为编码区序列。另外，提供的seqidno.8-11分别是slc20a2，pdgfrb，pdgfb，xpr1的野生型基因的编码区序列，供参比。本发明提供的基于pcr捕获测序的ibgc致病基因检测方法(参见图1)包括如下主要步骤：(1)4个致病基因的pcr捕获提取待测样本基因组dna，利用设计合成的pcr捕获引物组进行扩增；(2)二代测序解析基因突变谱将(1)中的多重pcr扩增产物进行二代测序，并通过信息分析找出突变位点；(3)突变位点的验证对于结果是已知突变的，则将原样本突变片段扩增后直接进行sanger测序验证；对于结果是新发现突变的，则根据情况设计引物将原样本突变片段扩增后直接进行sanger测序验证后，再通过sanger测序分析患者家系相关成员样本的基因型-表型共分离情况进行验证。其中，(1)中所述pcr捕获引物组是利用美国thermofisher公司的在线软件ionampliseqdesigner进行设计的，至少应该捕获所述所有7种突变形式其突变位点所在区域，结果显示至少需要seqidno.12-23这12条序列。优选地，可以针对slc20a2、pdgfrb、pdgfb和xpr1这4个基因所有外显子及其侧翼序列，使得目标基因覆盖率达100％，结果共获得了69对引物，其序列如表1所示。表1pcr捕获扩增引物组其中，(1)中进行的多重pcr扩增体系(20ul)如表2所示。5xionampliseqtmhifimix、2xionampliseqtm引物组和20xionampliseqtmsampleidpanel均购置于美国thermofisher公司。表2pcr捕获扩增体系成分添加量(ul)5xionampliseqtmhifimix42xionampliseqtm引物组1020xionampliseqtmsampleidpanel1浓度>20ng/ul的样本基因组dna0.5灭菌的nuclease-freewater4.5其中，(1)中所述的多重pcr扩增阶段和条件如表3所示：表3pcr捕获扩增阶段和条件其中，(2)中所述的pcr扩增捕获产物进行的二代测序和信息分析，有二代测序仪和配套分析软件则按照规范操作和分析进行，或可由商业化的高通量测序公司完成，最终需得到所检测样本的致病基因突变信息。其中，(3)中所述的已知突变指的是前述的7种突变形式(可详见表4)，所述新发现突变指的是这7种已发现突变外的其它突变形式。所述的原样本是指(1)中提取的待测样本基因组，除用于pcr捕获测序模板外剩余的部分；或者原检测样本再次提取的基因组dna。所述突变包括点突变、插入或缺失，但不限于此。其中，(3)中所述对于已知突变则将原样本突变片段扩增后直接进行sanger测序验证，是指利用特定的引物对扩增突变片段并进行sanger测序，验证是否同二代测序检出的突变结果一致；有sanger测序仪则按规范操作进行测序或可送商业化测序公司完成，最终需得到对应的扩增子序列。所述原样本突变片段扩增是指，对于slc20a2基因的c.799g>c突变，扩增引物对为seqidno.24-25；对于pdgfrb基因的c.3g>a和c.2209g>a突变，扩增引物对分别为seqidno.26-27和seqidno.28-29；对于pdgfb基因的c.220g>t和c.232c>t突变，扩增引物对分别为seqidno.28-29和seqidno.30-31；对于xpr1基因的c.490g>t和c.1708c>t突变，扩增引物对分别为seqidno.32-33和seqidno.34-35。其中，(3)中所述的患者家系相关成员必须包括ibgc患者和非ibgc患者。所述基因型-表型共分离是指基因型决定表型，由此两者符合共同对应的遗传学传递现象和规律。本发明提供的ibgc相关4个致病基因的7种突变基因类型，可以为该病致病机理的解析和药物开发、致病基因筛查和检测以及治疗方案的制定等提供依据和奠定基础；此外本发明提供的基于pcr捕获测序的致病基因检测方法，覆盖了4个致病基因的全部外显子，可以高效全面、快速准确地获取突变信息。附图说明图1是本发明ibgc致病基因检测方法流程图。图2-8是各种基因突变形式的sanger测序验证图谱突变位点局部。其中图2对应于实施例1中的1号患者，为slc20a2基因的c.799g>c突变，2号、3号和4号患者的图谱与其类似；图3、图4、图5、图6、图7、和图8分别对应于实施例1中的5、6、7、8、9和10号患者，分别为pdgfrb的c.3g>a、pdgfrb的c.2209g>a、pdgfb的c.220g>t、pdgfb的c.232c>t、xpr1的c.490g>t和xpr1的c.1708c>t；各图中的序列位点数字只代表对应扩增子，并不代表突变位点在实际基因中的位置。图9是实施例1中1号ibgc患者颅脑ct平扫影像。图10是实施例1中1号ibgc患者所在家系的遗传图谱。此图中正方形表示男性，圆形表示女性；箭头表示先证者，带斜线表示死亡，带问号表示未行颅脑ct检查个体；实心表示ibgc患病个体，空心表示正常个体；“+”表示slc20a2基因检出c.799g>c突变，“-”表示slc20a2基因未检出c.799g>c突变；i、ii和iii分别代表第1、2和3代，数字为成员编号。具体实施方式以下结合具体实施例，进一步阐述本发明。实施例1根据详细的病史和家族史调查情况以及规范的神经系统体格检查和相关辅助检查信息，选择所在医院10名ibgc患者在其签署了知情同意书的情况下，进行致病基因检测和分析。(1)4个致病基因的pcr捕获取患者肘静脉血10ml(edta抗凝)，用qiaampdnabloodminikit(德国qiagen公司)提取基因组dna，以其为模板采用表1的pcr捕获引物组按照表2配制扩增体系，并在常规pcr仪上按照表3条件进行反应。(2)二代测序解析基因突变谱将(1)中的多重pcr扩增产物送上海晶能生物技术有限公司进行二代测序，采用的是ionpgmtemplateot2200试剂盒进行乳液pcr，dynabeadsmyonestreptavidinc1磁珠进行阳性模板富集；以上两步反应均在iononetouch系统上进行。建库后采用ionpgmsequencingkit及iontorrent316v2芯片，在pgm测序仪上进行测序反应。采用pgm服务器自带分析软件对测序结果初步分析，再用千人基因组数据库、snp数据库和cosmic(version64)数据库等进行突变位点解析，结果如表4所示，共计有7种突变。表410例ibgc患者致病基因检测及验证结果(3)突变位点的验证根据所检出的突变，利用前述特定引物对将原样本突变片段扩增后直接进行sanger测序验证。采用takara的rtaq聚合酶扩增，其扩增体系详见表5，扩增引物对详见表6，pcr产物送上海生工生物工程股份有限公司进行sanger测序。sanger测序结果发现均与二代测序结果一致(表4)。表5扩增体系编号组份添加量110xpcrbuffer(mg+)2.5ul2dntpmix0.5ul3primer(50um)f0.25ul4primer(50um)r0.25ul5rtaq0.25ul6templatedna(30ng/ul)1ul7灭菌ddh2o20ul表6扩增引物对扩增条件为：94℃预变性3min；30个循环：94℃变性30s，60℃/65℃退火30s，72℃延伸30s；72℃终延伸5min。由于这7种突变均未被报道，因此收集这10例ibgc患者家系相关成员样本，再通过sanger测序突变片段。以s1号患者为例，如图9所示其为家系先证者iii4，无明显症状，颅脑ct平扫示双侧苍白球及小脑齿状核对称性大面积钙化。根据pgm数据可知，其slc20a2基因第799位的g突变为c；利用引物对seqidno.24-25扩增slc20a2基因c.799g>c突变片段，其直接sanger测序结果(如图2所示)，与二代测序结果一致。收集该家系中的其他成员资料，能获得该家系6个阳性成员，4个阴性成员的影像资料(如图10中所示)：即先证者iii4、ii1、ii3、ii8、iii2及iii7的颅脑ct平扫均显示双侧基底节区及小脑存在大片钙化灶，ii2、iii1、iii5、iii6颅内均无钙化灶。采集这10个家系成员的血液样本提取基因组dna，利用引物对seqidno.24-25扩增slc20a2基因c.799g>c突变片段，sanger测序显示6个阳性患者均存在该突变位点，而4个阴性成员均无该位点突变，即出现了基因型-表型的家系共分离现象，由此证明了slc20a2基因的c.799g>c突变是该家系的致病性突变。表4中其余9例患者，经过同样的分析均出现家系共分离现象(表4)。因此，通过本实施例获得了7种ibgc的致病基因，其序列对应信息详见表4。(4)致病基因制备和获取基于本发明新鉴定的ibgc的7种致病基因，为了开展致病机理机制、筛查检测方法和试剂盒开发、治疗药物研发等科研生产工作需要，申请人将突变所在的扩增子进行纯化，将其克隆到商业化质粒等载体中，之后将其导入大肠杆菌等宿主菌中，置于-80℃冰箱中保存。另一方面，由于本发明对于致病基因序列的公布，可以采用定点突变的方式由野生型基因制备得到，或通过序列合成的方式获取。实施例2另取与实施例1中不同的ibgc患者12例以及200例正常人血样进行分析，方法同实施例2，其结果如表7所示。表712例ibgc患者致病基因检测及验证结果对于表5中ibgc患者的突变均经过sanger测序验证了，所有突变基因均属于所述7种基因突变形式；而在200例正常人中，均不存在所述的突变，从而从正反两方面再次验证了本发明提供的ibgc致病基因，以及本发明提供的检测方法具有良好的可靠性和准确性。虽然本发明是通过优选的具体实施例进行说明，但是对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。序列表<110>福建医科大学附属第一医院<120>第220位点发生突变的人类ibgc致病基因pdgfb及其检测方法<141>2018-05-08<160>35<170>siposequencelisting1.0<210>1<211>3702<212>dna<213>智人(homosapiens)<400>1cgcttccggaagcgggcgactcgcagctccacgcgacgccgaggggctccgcgccgggac60cgggcgggtgctcggagtttcggggaccgcacgggaccgagggcaggaggagacatcaca120gctttcccagatcgggaggaaaaatatggaatgtgttttaccgctgactgaacacaacca180aatgaactgtcctgacagtagtttgcaaaccagcagctagcagtttgtccagcctctaac240attgtccagcactttccagagcaaactcactgtttacaagaactcttggccttacgaagt300ttataacctcaagctttgtttatttaaaatattcctgcaaaagaaaagtacccggcaccc360actttccaaaatggccatggatgagtatttgtggatggtcattttgggtttcatcatagc420tttcatcttggccttttctgttggtgcaaacgatgttgccaactcctttggtacagccgt480gggctctggtgtggtgaccttgaggcaggcatgcattttagcttcaatatttgaaaccac540cggctccgtgttactaggcgccaaagtaggagaaaccattcgcaaaggtatcattgacgt600gaacctgtacaacgagacggtggagactctcatggctggggaagttagtgccatggttgg660ttccgctgtgtggcagctgattgcttccttcctgaggcttccaatctcaggaacgcactg720cattgtgggttctactataggattctcactggtcgcaatcggtaccaaaggtgtgcagtg780gatggagcttgtcaagattgttgcttcttggtttatatctccactgttgtctggtttcat840gtctggcctgctgtttgtactcatcagaattttcatcttaaaaaaggaagaccctgttcc900caatggcctccgggcactcccagtattctatgctgctaccatagcaatcaatgtcttttc960catcatgtacacaggagcaccagtgctcggccttgttctccccatgtgggccatagccct1020catttcctttggtgtcgccctcctgttcgctttttttgtgtggctcttcgtgtgtccgtg1080gatgcggaggaaaataacaggcaaattacaaaaagaaggtgctttatcacgagtatctga1140cgaaagcctcagtaaggttcaggaagcacagtccccagtatttaaagagctaccaggtgc1200caaggctaatgatgacagcaccatcccgctcacgggagcagcaggggagacactggggac1260ctcggaaggcacttctgcgggcagccaccctcgggctgcatacggaagagcactgtccat1320gacccatggctctgtgaaatcgcccatctccaacggcaccttcggcttcgacggccacac1380caggagcgacggtcatgtgtaccacaccgtgcacaaagactcggggctctacaaagatct1440gctgcacaaaatccacatcgacaggggccccgaggagaagccagcccaggaaagcaacta1500ccggctgctgcgccgaaacaacagttacacctgctacaccgcagccatttgtgggctgcc1560agtgcacgccacctttcgagctgcggactcatcggccccagaggacagtgagaagctggt1620gggcgacaccgtgtcctactccaagaagaggctgcgctacgacagctactcgagctactg1680taacgcggtggcagaggcggagatcgaggcggaggagggcggcgtggagatgaagctggc1740gtcggagctggccgaccctgaccagccgcgagaggaccctgcagaggaggagaaggagga1800gaaggacgcacccgaggttcacctcctgttccatttcctgcaggtcctcaccgcctgttt1860cgggtcctttgctcacggcggcaatgacgtgagtaatgccatcggtcccctggtagcctt1920gtggctgatttacaaacaaggcggggtaacgcaagaagcagctacacccgtctggctgct1980gttttatggaggagttggaatctgcacaggcctctgggtctgggggagaagagtgatcca2040gaccatggggaaggacctcactcccatcacgccgtccagcggcttcacgatcgagctggc2100ctcagccttcacagtggtgatcgcctccaacatcgggcttccagtcagcaccacgcactg2160taaggtgggctcggtggtggccgtgggctggatccgctcccgcaaggctgtggactggcg2220cctctttcggaacatcttcgtggcctggttcgtgaccgtccctgtggctgggctgttcag2280cgctgctgtcatggctcttctcatgtatgggatccttccatatgtgtgagctgtcatggc2340tcttctcatgtatgggatccttccatatgtgtgatttgtcttcttccagctgcaaacagc2400taaagggatggtctggtgttggcgtgtgggagacatgtgtgctcgtgccgcacatacaca2460tcctggccgtgcacggctctctcatgaccagctctctgcctcccttccaggaggctccat2520cccacactgttcacccaggctgcggagactcaccttcccgagctaacttaactactgtac2580ataataatatgtattaaactggtatcgtggtgatataatgtggtgcagttacttatatat2640taaatatctattgtatccatagaataggcagcattatttcaaacatattcaagttgggag2700tggagatcattgcctagaagtcaatattcaataaatcttgtacataactatttcgatggc2760aaatgttaagccttctaaaaggaaagtgtagattggaaaatgattttttttccaaatgat2820gtttttgccttctaatatactgtaaggtaatgagcttcagaacaggcaacctgaccctgc2880agaggtcgcgtgctgtgggatgacagcgggacgggagctcacaagtgctttcactgaaga2940tttgttcatatactgtgtattgattgttgtgtaatatatcatcattgcttttgtaaatac3000gtaaaactgtaattttttaatggtgtgcttcccttatacttttttgatcagagaattttg3060gaaagtaccaaagaagcaggggaatcattggccagtgttacgttttcacattgtctgtct3120cccaccctcactgatcacgcctgccccagagcagtgtgtggcggtgacaccgtcacccag3180catgcgccacgccgtggctcccaccagcagtgccaccgccaccacaccccagatcccacc3240caccttgcagtggcctttccttgtcatcagagtagagaatgcacaggtgttggtgagggc3300gtgtggctgagcactacatgtcaagtccagagtcagtttctatcccaattctccctgcag3360cctgaagaacggatccttgtctccaatgtcagcacaaaggaggctttttctgtgctttga3420cattctagcacttcagggatgagagggagggagaatcctggatgctggatggagtatttc3480tctgaggcccacacaaagctggacacccccaggctctactccatcccatt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cialsequence)<400>22ccagctctgtcagtgcttagtg22<210>23<211>30<212>dna<213>人工序列(artificialsequence)<400>23caggtaaggcatatatattcaaagctagca30<210>24<211>21<212>dna<213>人工序列(artificialsequence)<400>24accacacctggcctcaacttc21<210>25<211>22<212>dna<213>人工序列(artificialsequence)<400>25tccctagcttctgggttttctc22<210>26<211>19<212>dna<213>人工序列(artificialsequence)<400>26tgggacgtggtggcttttg19<210>27<211>19<212>dna<213>人工序列(artificialsequence)<400>27acagagcccactggaaatc19<210>28<211>20<212>dna<213>人工序列(artificialsequence)<400>28ctctctgggttctaactgtc20<210>29<211>20<212>dna<213>人工序列(artificialsequence)<400>29tctatgaaatgggcacgagg20<210>30<211>19<212>dna<213>人工序列(artificialsequence)<400>30aggagggactgttctctgg19<210>31<211>20<212>dna<213>人工序列(artificialsequence)<400>31ctgtgatgactgaacacagg20<210>32<211>20<212>dna<213>人工序列(artificialsequence)<400>32aatctttgtccagccagctc20<210>33<211>19<212>dna<213>人工序列(artificialsequence)<400>33gggccagctaaatactgtg19<210>34<211>20<212>dna<213>人工序列(artificialsequence)<400>34tcgtaggacatgtgtgagtc20<210>35<211>19<212>dna<213>人工序列(artificialsequence)<400>35cctggccaacatggttttc19当前第1页12