CN114457087A - 早发冠心病相关的snp位点及其应用 - Google Patents

早发冠心病相关的snp位点及其应用 Download PDF

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CN114457087A
CN114457087A CN202210238216.6A CN202210238216A CN114457087A CN 114457087 A CN114457087 A CN 114457087A CN 202210238216 A CN202210238216 A CN 202210238216A CN 114457087 A CN114457087 A CN 114457087A
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阴赪茜
周璨
王子婧
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Beijing Anzhen Hospital
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Abstract

本发明属于生物技术领域，公开了一种与冠心病相关的基因突变、核酸及其应用，具体提供了一种AKAP9基因突变体和其应用。所提供的基因突变体与正常AKAP9基因相比，具有c.6406C>G突变。本发明所提供的基因突变是早发冠心病的一种新的相关的致病突变，通过检测该基因突变在生物样品中是否存在，可以有效地检测生物样品的供者是否患早发冠心病。进一步，本发明的AKAP9基因突变具有在早发冠心病早期预测的检测产品中的应用前景，本发明提供的早发冠心病相关的基因位点可能是冠心病的早期生物标志物，为进一步研究早发冠心病的遗传分子机制，探索早发冠心病早期防治的药物靶点提供了新的方向。

Description

早发冠心病相关的SNP位点及其应用
技术领域
本发明属于生物技术领域，具体涉及与冠心病相关的AKAP9基因突变体和其应用。
背景技术
冠状动脉粥样硬化性心脏病，简称冠心病，是指在冠状动脉血管发生粥样硬化病变，从而引起冠状动脉血管狭窄或闭塞，导致心肌缺血、缺氧甚至坏死的心脏病。随着社会经济的发展，冠心病发病率和死亡率逐年上升，2020年世界卫生组织(WHO)公布的全球十大死亡原因中，缺血性心脏病位居榜首，占世界总死亡人数的16％，成为全球最大杀手。值得一提的是，冠心病的发病年龄已经出现年轻化的趋势，发病群体年轻化进一步增加了我国的医疗和经济负担。
早发冠心病(premature coronary artery disease，PCAD)系指冠心病的一种早发分支，属于冠状动脉疾病，其划分标准为：男性≤55岁，女性≤65岁，且多伴有家族史。其中冠状动脉疾病(coronary artery disease，CAD)系指冠状动脉粥样硬化造成的管腔狭窄或阻塞，在此基础上发生的心肌急剧的、暂时的缺血与缺氧所引起的临床综合征。PCAD是一种特殊的冠心病类型，多无先兆症状，起病急，病死率高。随着社会经济的发展，心血管病危险因素流行趋势明显，冠心病发病率逐渐上升，且发病年龄也日渐年轻化；而预防和早期干预治疗可以明显降低早发冠心病的发病率和死亡率，改善预后，减轻医疗负担，是心血管内科工作中急需解决的临床难题之一。随着医学科学的不断发展，众多先进的新型检测诊断技术相继产生，进而使冠心病的诊断向安全快捷、无创、早期等方向逐渐发展。而对于冠心病的年轻发病人群来说，遗传因素的影响显然比环境因素更加重要。目前全基因组协会(GWA)已经确定了几个与PCAD风险相关的遗传基因组，而不同种族的遗传及基因组差异也导致与PCAD风险相关的基因组的不同。因此，通过考察对比多种族的遗传基因组，确定不同种族的特异性基因组及其共同的易感基因位点对PCAD的研究至关重要。
伴随着人类基因组测序计划完成和高通量测序技术的发展，医学领域的科研工作者对人类基因组有了更深入的认识，对疾病相关遗传变异、相关基因的功能及调控网络的认识也逐渐加深，高通量测序得到广泛应用。全外显子组测序(Whole Exome Sequencing，WES)技术是利用外显子捕获试剂盒捕捉整个外显子区域DNA，然后进行高通量测序，是目前基因组学研究中非常成熟的一部分。WES相较于全基因组测序(Whole Genome Sequencing，WGS)、全基因组关联分析(genome wideassociation studies，GWAS)具有对基因组编码区域的高深度测序，除大片段的缺失重复及单核苷酸多态性(SNP，single nucleotidepolymorphism)外，还能发现低频突变、罕见突变，识别范围更广，具有低成本、高效率的优势，是寻找疾病易感基因或位点特别是发现新易感基因或位点最常用的方法之一，可弥补GWAS不能筛选未知候选基因的缺陷。
PCAD发病急骤，风险高，不易早期发现，基于已知致病基因的遗传学诊断阳性率有限，难于做早期风险评估，亟待发现新的分子标志物。
发明内容
为了解决上述问题，本发明的第一个方面是提供一种与冠心病相关的AKAP9基因突变体和其应用，具体涉及基于全外显子测序技术筛选得出AKAP9基因突变体，与正常AKAP9基因(SEQ ID NO.1)相比，其SNP突变位点为6406C>G，即具有c.6406C>G突变。发明人发现AKAP9的基因c.6406C>G突变的AKAP9的基因突变体(SEQ ID NO.2)与冠心病密切相关，从而可以通过检测该基因突变在生物样品中是否发生，有效地检测生物样品的供者是否患冠心病。
本发明的第二个方面是提供一种核酸，与正常AKAP9基因相比，所述核酸具有c.6406C>G突变。发明人发现AKAP9的基因c.6406C>G突变与冠心病密切相关，从而可以通过检测该核酸在生物样品中是否发生，有效地检测生物样品的供者是否患冠心病。
本发明的第三个方面是提供一种多肽，与正常AKAP9基因表达的多肽的氨基酸序列相比，所述多肽的氨基酸序列具有p.Q2136E突变。该突变与冠心病密切相关，从而可以通过检测该多肽突变在生物样品中是否发生，有效地检测生物样品的供者是否患冠心病。
本发明的第四个方面是检测上述的基因突变或核酸或多肽的试剂在制备试剂盒或者设备中的用途，所述试剂盒或者设备用于诊断冠心病；
优选的，所述的试剂包括特异性针对所述基因突变、所述核酸和所述多肽的至少之一的抗体、探针、引物以及质谱检测试剂的至少之一。
优选的，所述的试剂盒包括检测早发冠心病易感性SNP位点AKAP9_c.6406C>G_p.Q2136E的基因、核酸或多肽是否发生突变的试剂。
优选的，所述试剂盒可以为采用本领域已知的任何技术检测SNP的试剂，只要其能够检出样本中易感SNP位点AKAP9_c.6406C>G_p.Q2136E是否存在突变，所述试剂盒中包括检测所述的基因突变的试剂，和/或检测所述的核酸的试剂，和/或检测所述的多肽的试剂。其包括但不限于下面列举的各实施方式。
在第一实施方式中，该试剂盒包括利用测序法检测样本中易感SNP位点AKAP9_c.6406C>G_p.Q2136E位点G等位基因存在的试剂。测序法是本领域所公知的技术，所需的引物等试剂，本领域普通技术人员均可根据需要自行选择(参见ABI、Beckman等公司测序仪相关使用说明)，在此不再赘述。利用该试剂盒，通过测序法可以直接测得样本中易感SNP位点AKAP9_c.6406C>G_p.Q2136E的序列，从而判断其是否携带相应位点等位基因的变异，进而判断其早发冠心病的易感性。
在第二实施方式中，所述试剂盒包括利用Taqman探针SNP检测法检测样本中易感SNP位点AKAP9_c.6406C>G_p.Q2136E基因型的试剂。其中所用Taqman探针为针对易感SNP位点AKAP9_c.6406C>G_p.Q2136E设计的探针，该探针可以使用试剂公司提供的探针；也可使用软件自行设计，如PREMIERBiosoft公司的BeaconDesigner 7.5。
在第三实施方式中，所述试剂盒为利用PCR-单链构象多态性法检测样本中易感SNP位点AKAP9_c.6406C>G_p.Q2136E基因型的试剂盒。该试剂盒中包括用于扩增易感SNP位点AKAP9_c.6406C>G_p.Q2136E的引物，PCR试剂、对照样本和检测构象的电泳所需试剂。所述电泳优选非变性聚丙烯酰胺凝胶电泳。对照样本中包括易感SNP位点AKAP9_c.6406C>G_p.Q2136E CC纯合子的阴性对照样本和位点GG纯合子的阳性对照样本中的至少一个，此外还可以包括也可以不包括杂合子相应杂合子的对照样本。优选同时包括上述三类对照样本。待测样本扩增产物与对照样本的扩增产物同时电泳，比较其电泳结果可以得出待测样本中是否携带相应等位基因变异的检测结果。
进一步地，本发明提供了所述SNP位点在筛选药物的生物模型中的用途，其中所述生物模型选自下列至少之一：
(1)上述的基因突变；
(2)上述的核酸；
(3)表达上述的多肽；
优选的，所述生物模型为细胞模型或者动物模型。
进一步地，本发明提供了特异性改变以上所述的基因突变或者核酸的试剂在制备药物中的用途，所述药物用于治疗冠心病；
优选地，所述冠心病为早发冠心病。
更进一步地，本发明提供了所述的SNP位点在制备诊断早发冠心病装置中的应用。
优选的，所述诊断装置是测序芯片。
再进一步地，本发明提供一种筛选与早发冠心病相关的SNP位点的方法，所述方法包括以下步骤：
(1)提取早发冠心病患者和正常对照的外周血样本的DNA；
(2)将DNA进行超声片段化，随机打断成长度为180-280bp的片段，经末端修复和加A尾后在片段两端分别连接上接头制备DNA文库；
(3)采用Agilent的液相芯片捕获系统，对人的全外显子区域DNA进行高效富集，然后在Illumina平台上进行高通量双端测序，每端读长为150bp；
(4)外显子测序后，进行数据质量评估，对测序错误率、数据量和比对率进行统计，并将有效测序数据以GRCh37/hg19为参考序列或参考基因组通过Burrows-WheelerAligner(BWA)进行序列比对，使用SAMtools对比对结果进行排序，再用Sambamba标记重复reads，最后利用重复标记后的比对结果进行覆盖度、深度的统计。
(5)在上述比对结果的基础上利用GATK4识别SNP，InDel以及CNV，利用ANNOVAR软件对上述位点进行注释，其中包括dbSNP数据库、千人基因组计划和其他已有的数据库的注释信息，注释内容涵盖变异的位置信息，类型，保守型预测，主要步骤包括碱基质量重矫正(BQSR)，局部重比对(local realignment)以及基于单倍型的基因分型；生成的变异位点文件(VCF)进一步通过ANNOVAR软件进行基因组信息以及功能注释。
(6)对变异检测的结果，通过基于人群频率(参考频率包括1000genomes，ExAC，genomAD)、变异的功能有害性预测、物种保守性分析以及所在基因的组织表达水平进行筛选；最后对候选的变异位点结合临床表型进行遗传解读，包括遗传位点数据库检索(数据库包括HGMD，OMIM，ClinVar)，家系疾病共分离分析，以及ACMG致病性评级。最终发明人筛选出一个早发冠心病易感性SNP位点：AKAP9_c.6406C>G_p.Q2136E。
有益效果：
本发明通过发现了一个早发冠心病易感SNP位点并研究了其在早发冠心病早期预测的应用前景，本发明提供的早发冠心病相关的基因位点可能是早发冠心病的早期生物标志物，为进一步研究早发冠心病的遗传分子机制，探索早发冠心病早期防治的药物靶点提供了新的方向，也为个体化治疗的顺利实施提供了依据。
附图说明
图1为入组中国人早发冠心病突变位点与疾病共分离的家系图
图2为AKAP9在主动脉和心脏中的高度表达柱状图
图3为Gln2136Glu(Q2136E)位点在家系的全外显子测序数据中IGV图
图4为早发冠心病家系中AKAP9基因Q2136E突变的Sanger测序验证
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1、样本搜集
在首都医科大学附属北京安贞医院就诊的中国人早发冠心病家系(图1)，家系的9名成员中6名为冠心病患者(S02,S03,S06,S07,S08,S09；其中S03因急性心肌梗死去世)，3名为健康对照(S01,S04,S05)，除1人因急性心肌梗死去世以外，纳入研究共计8人。该家系为非常典型的早发冠心病家系，两代人中均有早发冠心病发病，发病年龄明显低于早发冠心病的年龄诊断标准，5名患者中诊断为急性心肌梗死2人，不稳定型心绞痛3人，5名患者均接受不同次数的介入治疗或心脏搭桥手术，且排除糖尿病、高血脂、高血压、吸烟、饮酒等传统危险因素。
入选标准：①冠心病诊断标准为至少有1支主要冠状动脉管径狭窄≥50％，诊断为冠心病；②冠心病发生时男性低于55岁，女性低于65岁；诊断为早发冠心病。
同一家系中未发病且无冠脉狭窄的健康成员为健康对照组。
所有患者均须签署知情同意书，本实验方案得到首都医科大学附属北京安贞医院医学伦理委员会的批准。
标本采集：符合入选标准的患者和健康对照者，通过桡静脉抽取外周血10mL，并置于EDTA抗凝管中，冻存于-80℃超低温冰箱。
实施例2、全外显子测序
DNA提取及检测：使用NEB
Figure BDA0003543200280000031
UltraTM DNA Library Prep Kit for
Figure BDA0003543200280000032
自外周血样本提取基因组DNA；运用琼脂糖凝胶电泳分析DNA降解程度以及是否有RNA、蛋白质污染；使用Qubit3.0对DNA浓度进行精确定量，其中含量在0.6ug以上的DNA样品被用来建库。
建库捕获：将基因组DNA经Covaris破碎仪随机打断成长度为180-280bp的片段，经末端修复和加A尾后在片段两端分别连接上接头制备DNA文库。带有特异index的文库pooling后与生物素标记的探针进行液相杂交，再使用带链霉素的磁珠将基因上的外显子捕获下来，经PCR扩增后进行文库质检，合格即可进行测序。
文库质量检测：文库构建完成后，先使用Qubit2.0进行初步定量，随后使用NGS3K/Caliper对文库的insert size进行检测，insert size符合预期后，使用qPCR方法对文库的有效浓度(3nM)进行准确定量，以保证文库质量。
上机测序：库检合格后，根据文库的有效浓度及数据产出需求运用Illumina平台进行测序，所用测序模式为PE150(Pairend 150bp)，即高通量双端测序，每端各测150bp。
获得原始测序序列(Sequenced Reads)后，在有参考序列或参考基因组(GRCh37/hg19)的情况下，进行信息分析流程，包括以下三个部分：
(1)测序数据质量评估：对测序错误率、数据量和比对率进行统计，评估建库测序是否达到了标准，符合标准则进行后续分析，否则需重新建库或加测。经过去除接头及低质量reads(单端测序read中N(N表示无法确定碱基信息)的比例大于10％，或单端测序read中含有的低质量(低于5)碱基数超过该条read长度比例的50％)进行测序数据过滤；通过Phred值(Phred score,Qphred)进行测序错误率分布检查；有效测序数据通过Burrows-Wheeler Aligner(BWA)比对到参考基因组(GRCh37/hg19)，得到BAM格式的最初的比对结果，然后用SAMtools对比对结果进行排序，再用Sambamba标记重复reads，最后利用重复标记后的比对结果进行覆盖度、深度的统计；在测序reads能达到95％以上的比对率、位点的碱基覆盖深度(read depth)达到10X以上时，该位点处检测出的SNP比较可信。
(2)变异检测与注释：在上述比对结果的基础上利用GATK4识别SNP，InDel以及CNV，利用ANNOVAR软件对上述位点进行注释，其中包括dbSNP数据库、千人基因组计划和其他已有的数据库的注释信息，注释内容涵盖变异的位置信息，类型，保守型预测，主要步骤包括碱基质量重矫正(BQSR)，局部重比对(local realignment)以及基于单倍型的基因分型；生成的变异位点文件(VCF)进一步通过ANNOVAR软件进行基因组信息以及功能注释。
(3)变异位点筛选：对变异检测的结果，通过基于人群频率(参考频率包括1000genomes，ExAC，genomAD)、变异的功能有害性预测、物种保守性分析以及所在基因的组织表达水平进行筛选；最后对候选的变异位点结合临床表型进行遗传解读，包括遗传位点数据库检索(数据库包括HGMD，OMIM，ClinVar)，家系疾病共分离分析，以及ACMG致病性评级。
结果：为了确定导致中国汉族人早发冠心病的基因组突变，发明人对招募的8个家庭成员进行了全外显子组测序。全外显子测序数据分析结果表明，在5名患者(S02,S06,S07,S08,S09)和1名携带者成员(S05)中发现了AKAP基因(SEQ ID NO.1)的非同义突变Q2136E(Gln2136Glu)(携带者年龄为37岁，可能尚未达到发病年龄)，而在2名健康对照成员(S01,S04)中则不存在(图1)。经查阅genomAD和ExAC数据集的千人基因组中没有该突变。且Q2136在人、恒河猴、小鼠、鸡和X_tropicalis中高度保守。从GTEX数据集检索到的表达水平表明，AKAP9在主动脉和心脏中高度表达(图2)。Gln2136Glu(Q2136E)位点在家系的全外显子测序数据中，该变异位点与家系中的患病成员疾病共分离，在患病的S06，S07，S08中均出现，但在不患病的S04中缺失。
表1早发冠心病SNP位点信息
Gene Chr,Pos AA_change
AKAP9 chr.7,91947267 AKAP9_c.6406C>G_p.Q2136E
注：Gene，基因名称；Chr，染色体；Pos，SNP位置；AA_change，氨基酸改变
实施例3、利用Sanger测序验证突变位点
测序模板DNA提取及检测：使用磁珠法自外周血样本提取基因组DNA：取200ul抗凝血，加入等体积PBS缓冲液中，12000g/min离心五分钟；弃上清，加入440ul溶液I，10ul蛋白酶K，56℃1h；加入等体积溶液II，5ul磁珠，混匀后室温静置5min；取上清，加入70％酒精，去上清；重复前一步骤；吹干后加入25ul TE缓冲液，-20℃保存。运用琼脂糖凝胶电泳分析DNA模板质量，检测无问题后进行PCR扩增。
引物设计：采用Primer Premier5.0软件设计合成引物，引物序列如下：
正向引物：5‘-GTAGATATCCAAAATGAG-3’(SEQ ID NO.3)
反向引物：5‘-AATGGTTACAATGTCAACAG-3’(SEQ ID NO.4)
PCR扩增：PCR反应体系如表2所示。扩增程序为：
Figure BDA0003543200280000041
2-4进行10个循环
5-7进行27个循环
表2反应体系
Figure BDA0003543200280000051
测序产物纯化：测序产物加入等体积乙醇混匀，静置后离心，弃除上清液；加入75％(或85％)乙醇漂洗，离心后弃除上清液，晾干；加入HI-DI，溶解后95度变性。测序产物纯化后，配制测序反应体系，进行上机测序。
Sanger法DNA测序：测序反应体系：ddH2O，DNA模板(质粒DNA、PCR产物等)，反应缓冲液(5Xbuffer)，反应缓冲液(2.5Xbuffer)，DNA聚合酶，引物(单向)，dNTP，ddNTP(四种，分别用4种不同荧光基团标记)；测序反应仪器型号：ABI3730XL。
结果：通过Sanger测序证实Q2136E突变存在于S06，S07，S08患者中，而非S04健康对照(图3)。进一步验证了该突变位点和早发冠心病的相关性。
实施例4、用于预测早发冠心病早期诊断试剂盒的制作
组装本发明所述的用于早发冠心病预测的试剂盒：
所述试剂盒包括检测AKAP9_c.6406C>G_p.Q2136E位点是否发生突变的试剂。
所述试剂包括用于扩增AKAP9_c.6406C>G_p.Q2136E位点的特异性引物，该引物可以采用软件来设计，如使用Primer5、Oligo6等；所述试剂还可以有相应PCR技术所需的常用试剂，如：dNTPs，MgCl2，双蒸水，Taq酶等，这些常用试剂都是本领域技术人员熟知的，另外还可以有标准品和对照(如确定基因型的标准品和空白对照等)。此试剂盒的价值在于只需要外周血而不需要其他组织样品，通过最精简和特异的引物对检测SNP，再通过SNP谱辅助判断早发冠心病风险，不仅稳定，检测方便，且精确，大大提高疾病诊断的敏感性和特异性，因此将此试剂盒投入实践，可以帮助指导诊断和更有效的个体化治疗。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 首都医科大学附属北京安贞医院
<120> 早发冠心病相关的SNP位点及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11724
<212> DNA
<213> Homo sapiens
<400> 1
atggaggacg aggagagaca gaagaagctg gaggccggca aagccaagct tgcccagttt 60
cgacaaagaa aagctcagtc ggatgggcag agtccttcca agaagcagaa aaaaaagaga 120
aaaacgtcaa gcagtaaaca tgatgtgtca gcacaccatg atttgaatat tgatcaatca 180
cagtgtaatg aaatgtacat aaatagttct cagagagtag aatcaactgt gattcctgaa 240
tctacaataa tgagaactct acatagtgga gaaataacca gtcatgagca gggcttctct 300
gtggaactgg aaagtgaaat ttcaaccaca gcagatgact gcagttcaga ggtaaatggt 360
tgcagttttg tgatgagaac aggaaagcct acaaatttat taagggaaga agaatttggt 420
gttgatgatt cttattctga acaaggagca caagacagtc cgactcatct agagatgatg 480
gaaagtgagt tggctgggaa gcagcatgag attgaagagc taaacagaga gctggaagaa 540
atgagggtta cctatgggac tgaaggactg cagcagttac aagaatttga agctgccatt 600
aaacaaagag atggcattat aacccagctc actgctaatt tacaacaagc aagaagagaa 660
aaggatgaga caatgagaga atttttagag ttgacagaac agagtcaaaa attacagatt 720
caatttcagc aattacaggc tagtgaaact ctgagaaaca gcactcatag tagcacagct 780
gcagacttac tacaagccaa acaacagatc ctcactcatc aacagcagct tgaagaacaa 840
gaccacttat tagaagatta tcagaaaaag aaagaagact tcacaatgca aattagtttc 900
ttgcaagaga aaattaaagt atatgaaatg gaacaagata aaaaagtaga aaactcaaat 960
aaagaagaaa tacaggaaaa ggagacaatc attgaagaat taaacacaaa aataatagaa 1020
gaagaaaaga aaactcttga gctaaaggat aaattaacaa ctgctgataa attactagga 1080
gaattacaag aacagattgt gcaaaagaac caagaaataa aaaacatgaa attagagctg 1140
actaattcta agcaaaaaga aagacagtct tctgaagaaa taaaacagtt aatggggaca 1200
gtcgaagaac ttcagaagag aaatcataaa gacagccagt tcgaaactga tatagtacaa 1260
cgaatggaac aagaaacaca aagaaagtta gaacaactcc gggcagagct ggatgagatg 1320
tatgggcagc agatagtgca aatgaaacaa gaattaataa gacaacacat ggcacagatg 1380
gaggaaatga aaacacggca taagggagaa atggagaatg ctttaaggtc atattcaaat 1440
attacagtta atgaagatca gataaagtta atgaatgtgg caataaatga actgaatata 1500
aaattgcaag atactaactc tcaaaaggaa aaactcaagg aagaactagg actaatttta 1560
gaagaaaagt gtgctctaca gagacagctt gaagaccttg ttgaagaatt gagcttttca 1620
agggaacaga ttcagagagc tagacagaca atagctgaac aagaaagtaa acttaatgaa 1680
gcacataagt cccttagtac agtggaagat ttgaaagctg agattgtttc tgcatctgaa 1740
tccagaaagg aactagaatt aaaacatgaa gcagaagtta caaattacaa gataaaactt 1800
gaaatgttag aaaaagaaaa gaatgctgtg ttagacagaa tggctgaatc acaagaagct 1860
gaattagaga ggctgagaac acagcttcta tttagtcacg aagaagagct ttccaaactg 1920
aaggaagatt tagaaattga acatcgaata aatattgaaa aacttaaaga taatttaggc 1980
attcactata aacagcagat agatggttta cagaatgaaa tgagtcaaaa gatagaaacc 2040
atgcagtttg aaaaggacaa tttgataact aagcagaatc aattaatttt ggaaatttca 2100
aagctaaaag atttacagca gtctcttgta aattcaaagt cagaagaaat gactcttcaa 2160
atcaatgaac ttcaaaaaga aattgaaata ctcagacaag aagaaaaaga aaagggtaca 2220
cttgaacaag aagttcaaga attacaactt aaaacagaat tgttagaaaa acagatgaag 2280
gaaaaagaga atgatcttca agaaaaattt gcacaacttg aagcagagaa tagcattctt 2340
aaagatgaaa agaaaaccct tgaagacatg ttgaaaatac atactcctgt tagccaagaa 2400
gaaagattga ttttcttaga ctccattaag tccaaatcca aagactctgt gtgggaaaaa 2460
gaaatagaaa tacttataga ggaaaatgag gacctcaaac aacaatgtat tcagctaaat 2520
gaagagattg aaaagcaaag gaacactttt tcatttgctg aaaaaaactt tgaagttaac 2580
tatcaagagt tacaagagga gtatgcttgc cttctcaaag taaaagatga tttagaagac 2640
agtaaaaata aacaggaatt agagtataaa agtaaactta aagcacttaa tgaagagctt 2700
catttgcaaa gaataaatcc aactacagtg aaaatgaaaa gttctgtctt tgatgaagac 2760
aaaacttttg tagcagaaac attggaaatg ggtgaggttg ttgaaaagga tacaacagaa 2820
ctcatggaaa aacttgaggt aaccaagcga gagaaattag agctgtcaca gagactgtct 2880
gatctttctg aacaattgaa acagaaacat ggtgagatta gttttctaaa tgaagaagtt 2940
aaatctttaa agcaagagaa agaacaagtt tcattgagat gtagagagct agaaatcatt 3000
attaaccaca acagggcaga aaatgtacag tcatgtgata ctcaagtaag ctctttatta 3060
gatggagttg tgaccatgac aagcaggggt gctgaaggat cagtttctaa agtaaataaa 3120
agttttggtg aagaatcaaa aataatggtg gaagataaag tttcttttga aaatatgact 3180
gttggagaag aaagtaagca agaacagttg attttggatc acttaccatc tgtaacaaag 3240
gaatcatcac ttagagcaac tcaaccaagt gaaaatgata aacttcagaa agaactcaat 3300
gtacttaaat cagaacagaa tgatttaagg ctacagatgg aagcccaacg catttgcctc 3360
tctctggttt attcaactca tgtggatcag gttcgtgaat atatggaaaa tgaaaaagat 3420
aaagctcttt gcagtcttaa agaagagctt atttttgctc aagaggaaaa gatcaaggaa 3480
cttcagaaaa tacaccagtt agaactacag actatgaaaa cacaagaaac aggtgatgaa 3540
ggaaagcctt tacatctgct cattggaaaa cttcaaaagg cagtgtctga agaatgttct 3600
tattttttac agactttatg cagtgtcctt ggtgaatatt atactcctgc tttaaaatgt 3660
gaagtaaatg cagaagacaa agagaattct ggtgattaca tttctgaaaa tgaagatcca 3720
gaattacaag attatagata tgaagttcaa gactttcaag aaaatatgca cactcttctc 3780
aacaaagtaa cagaagaata caacaaactc ttggtacttc aaacacgact aagcaagatc 3840
tggggacagc agacagatgg tatgaaactt gaatttggag aagaaaacct tccaaaagag 3900
gaaacagagt ttttatcaat ccattctcag atgaccaatt tggaagacat tgatgtcaat 3960
cataaaagca agttatcttc tctgcaagat cttgaaaaaa ctaaacttga agaacaagtt 4020
caagaattag aaagcctcat atcctctttg cagcaacagt tgaaagaaac tgaacaaaac 4080
tatgaggcag agatccactg tttacagaag aggcttcaag ctgttagtga gtccacggtt 4140
ccgccaagct tacctgttga ttcggtggta attacagaat ctgatgcaca gagaacaatg 4200
taccctggaa gttgtgtgaa aaagaatatt gatggtacaa tagagttttc tggtgaattt 4260
ggagtgaaag aggaaacaaa tatcgttaag ttgcttgaaa aacaatacca agaacaatta 4320
gaagaagaag tagctaaggt tattgtgtca atgagtatag catttgctca acaaactgaa 4380
ctgtctagaa tatctggggg aaaagaaaat actgcatcat caaagcaagc acatgctgtg 4440
tgtcagcaag aacaacatta ttttaatgaa atgaaattat cacaggatca aattggtttt 4500
cagacttttg agacagtgga tgtgaaattt aaagaagaat ttaaaccact tagtaaagag 4560
ttaggagaac atggaaagga aattttatta tcaaatagtg atccccatga tataccagaa 4620
tcaaaggact gtgtgctgac tatttcagaa gaaatgttct ccaaagataa aacatttata 4680
gttagacagt ctattcatga tgagatttca gtgtcaagca tggatgcttc tagacaacta 4740
atgttgaatg aagaacagtt ggaagatatg agacaggaac ttgtacgaca ataccaagaa 4800
catcaacagg caacggaatt gttaaggcaa gcacatatgc ggcaaatgga gagacagcga 4860
gaagaccagg aacagctaca agaagagatt aagagactta atagacaatt agcccagaga 4920
tcctccatag ataatgaaaa cctggtttca gagagagaga gggtgctttt agaggagctg 4980
gaagcactaa agcagctgtc tttagctgga agagagaagc tgtgttgtga gctgcgcaac 5040
agcagtacgc aaacacagaa tggaaatgaa aaccaaggag aagttgaaga acaaacattt 5100
aaagaaaagg aattagacag aaaacctgaa gatgtgcctc ctgagatttt gtctaatgaa 5160
aggtatgcac tccagaaagc taataataga cttttgaaga tcctcttaga agttgtaaag 5220
acaacagcag ctgttgaaga aacaattggt cgccatgtcc ttgggattct agatagatct 5280
agtaaaagcc agtcatctgc cagcctaatt tggaggtcag aagcagaggc atctgtaaag 5340
tcatgtgtcc atgaggaaca tacaagagtt acagatgaat ccattccctc ttattctgga 5400
agtgatatgc caagaaatga cattaacatg tggtcaaaag taactgagga aggaacagag 5460
ctgtcacaac gacttgtgag gagtggtttt gctggaactg aaatagaccc tgaaaatgaa 5520
gaacttatgc tgaacattag ctctcgacta caagcagcag ttgaaaaact cctagaagcc 5580
ataagtgaaa ctagcagtca gcttgaacat gcgaaagtga cacagacaga gttgatgcgt 5640
gagtcattta gacagaaaca agaagcaaca gagtccctta agtgccaaga ggaacttcga 5700
gagcgccttc atgaggagtc cagggccaga gaacagctag ctgtggagct cagtaaggct 5760
gagggcgtca ttgatggcta tgcagatgaa aaaactcttt ttgaaaggca aattcaggaa 5820
aaaactgata taatagatcg tcttgagcag gagttgttat gtgcaagtaa caggttgcaa 5880
gaattggagg cagagcaaca gcagatccaa gaagaaagag aattactgtc cagacaaaag 5940
gaagctatga aagcagaggc aggcccagtt gaacaacaat tactacagga gacagaaaaa 6000
ttaatgaagg aaaaactaga agtacaatgt caagctgaaa aagtacgtga tgaccttcaa 6060
aaacaagtga aagctctaga aatagatgtg gaagaacaag tcagtaggtt tatagagctg 6120
gaacaagaaa aaaatactga actaatggat ttaagacagc aaaaccaagc attggaaaag 6180
cagttagaaa aaatgagaaa atttttagat gagcaagcca ttgacagaga acatgagaga 6240
gatgtattcc aacaggaaat acagaaacta gaacagcaac ttaaggttgt tcctcgattc 6300
cagcctatca gtgaacatca aactagagag gttgaacagt tagcaaatca tctgaaagaa 6360
aaaacagaca aatgcagtga gcttttgctc tctaaagagc agcttcaaag ggatatacaa 6420
gaaaggaatg aagaaataga gaaactggag ttcagagtaa gagaactgga gcaggcgctt 6480
cttgtgagtg cagatacttt tcaaaaggta gaggaccgaa aacactttgg agctgtagaa 6540
gctaaaccag aattgtccct agaagtacaa ttgcaggctg aacgagatgc catagacaga 6600
aaggaaaaag agattacaaa cttagaagag caattagaac agtttagaga agaactggaa 6660
aataagaatg aagaagttca acaattacat atgcaattag aaatacagaa aaaggaatct 6720
actacccgcc tacaagaact tgaacaggaa aacaaattat ttaaggatga catggagaaa 6780
ctgggacttg ccataaagga atctgatgcc atgtctactc aagaccaaca tgtgctattt 6840
gggaaatttg ctcaaataat acaggaaaaa gaggtagaaa ttgaccaatt aaatgaacaa 6900
gttacgaaac tccagcagca acttaaaatt acaacagata acaaggttat tgaagaaaaa 6960
aatgaactga taagggatct tgaaacccaa atagaatgtt tgatgagtga tcaagaatgt 7020
gtgaagagaa atagagaaga agaaatagag cagctcaatg aagtgattga aaaacttcaa 7080
caggaattgg caaatattgg acagaagaca tcaatgaatg ctcattccct ctcagaagaa 7140
gcagacagtt taaaacatca attggatgtg gttatagctg aaaagctggc cttggaacag 7200
caagtagaaa ccgctaatga agaaatgacc ttcatgaaaa atgtacttaa agaaaccaat 7260
tttaaaatga atcagctaac acaggaatta ttcagcttaa agagagaacg tgaaagtgtg 7320
gaaaagattc aaagcatacc agagaatagt gttaacgtgg ctatagatca tctgagcaaa 7380
gacaaacctg aactagaagt agtccttaca gaggatgctc ttaaatccct agaaaatcag 7440
acatacttca aatcttttga agaaaatggc aaaggttcca taattaattt ggaaacaagg 7500
ttgctacaac ttgagagcac tgttagtgca aaggacttag aacttaccca gtgttataaa 7560
caaataaaag acatgcaaga acaaggccag tttgaaacag aaatgcttca aaagaagatt 7620
gtaaacctac agaaaatagt tgaagaaaaa gtggctgctg ctcttgtcag tcaaatccaa 7680
cttgaggcag ttcaggaata tgcaaaattc tgtcaagata atcaaacaat ttcatcagaa 7740
cctgaaagaa caaatattca gaatttaaat caactaagag aagatgagtt ggggtcagat 7800
atatcagcat taaccttgag aatatcagaa ttagaaagcc aggttgttga aatgcatact 7860
agtttgattt tagaaaaaga acaagtagaa attgcagaaa aaaatgtttt agaaaaagaa 7920
aagaagctgc tagaactaca gaagctattg gagggcaatg agaaaaaaca gagagagaaa 7980
gaaaagaaaa gaagccctca agatgttgaa gttctcaaga caactactga gctatttcat 8040
agcaatgaag aaagtggatt ttttaatgaa ctcgaggctc ttagagctga atcagtggct 8100
accaaagcag aacttgccag ttataaagaa aaggctgaaa aacttcaaga agagcttttg 8160
gtaaaagaaa caaatatgac atctcttcag aaagacttaa gccaagttag ggatcacctc 8220
gcagaggcaa aagagaaatt gtccatttta gaaaaagaag atgagactga ggtacaagaa 8280
agcaaaaagg cctgcatgtt tgagccactt cctataaaac tgagtaagag cattgcatcc 8340
cagacagatg ggactctgaa gatcagtagc agcaatcaga ctccacaaat tcttgttaaa 8400
aatgcaggaa tacaaattaa tttacagagt gaatgttcct cagaagaagt tactgaaata 8460
atcagtcagt ttactgaaaa aattgagaag atgcaagaac tacatgctgc tgaaattttg 8520
gacatggaat ccagacatat ttcagaaact gaaaccttaa agagggaaca ctatgttgcc 8580
gttcagttac tgaaagagga atgtggtacc ttgaaggcag tgatacagtg tctgagaagt 8640
aaagagggat cctcaattcc tgagctagca cattctgatg cttaccagac tagagaaata 8700
tgctccagtg attctggatc agactggggt cagggaattt atcttacaca cagtcaggga 8760
tttgacatag catcagaagg ccgaggagaa gaaagtgaaa gtgcaacaga ttcctttcca 8820
aagaaaataa agggattact gagagctgtc cataatgaag gcatgcaggt gctttctctc 8880
actgagtctc cctatagtga tggagaggac cattctattc agcaggtttc agaaccttgg 8940
ctagaagaga gaaaagctta catcaataca atctcatctc taaaggattt aattacaaag 9000
atgcaactgc aaagagaagc cgaggtttat gatagttctc aatctcatga gagcttctca 9060
gactggcgag gtgaactact gcttgccctt caacaagttt tcttagaaga gcgtagtgtt 9120
ttactagcag catttcggac ggagctgaca gctctaggta ctacagatgc agttggttta 9180
ctaaactgtt tggaacagag aatacaagaa cagggtgttg aatatcaagc agctatggaa 9240
tgcctccaga aagcagatag aaggagtttg ttatctgaaa ttcaggcact gcatgcacaa 9300
atgaatggta ggaaaattac tctgaaaaga gaacaagaga gtgagaaacc aagccaagaa 9360
ctcttggaat ataatataca gcagaagcag tctcaaatgc tggagatgca agtggagctc 9420
agcagtatga aagacagagc aacggaactg caggagcagc tgagttctga gaaaatggtg 9480
gttgctgaac tgaagagtga gcttgcacaa actaaattgg aactagaaac aacactcaag 9540
gcacagcata aacacctaaa agaattggag gctttcaggt tggaagttaa agataagaca 9600
gatgaagtac atttgcttaa tgacacatta gcaagtgaac agaaaaaatc aagagagctc 9660
cagtgggctt tggagaaaga gaaagccaag ttgggacgca gtgaagaacg ggataaagaa 9720
gaacttgagg atctgaagtt ttcacttgag agtcagaaac aaaggaatct tcagctaaat 9780
ctacttttgg aacaacagaa acaactactg aacgaatccc agcaaaaaat agaatcacag 9840
agaatgctat atgatgccca gttgtcagaa gaacaaggtc gaaacttaga gcttcaggta 9900
cttcttgaat ctgagaaagt tcgaattcgg gaaatgagta gtaccctaga tagggagcgg 9960
gaattgcacg cacagctgca gagcagtgat ggtactggac agtctcggcc acccttgccc 10020
tcagaggacc tactgaaaga gctgcagaaa cagctagagg aaaaacacag tcgcatagta 10080
gaattgttaa atgagactga aaaatataaa ctggattctt tgcaaacacg acagcaaatg 10140
gaaaaagata ggcaggttca caggaaaaca ctgcagacag aacaggaggc caacactgag 10200
ggacagaaaa aaatgcatga gctccagtcc aaagtggaag atcttcagcg ccagctggaa 10260
gagaaaagac aacaagttta taagttagac cttgaaggac agcgactaca aggaatcatg 10320
caggaattcc agaagcaaga actagaacga gaagaaaaac gagaaagtag aagaattctg 10380
tatcagaacc ttaatgagcc aaccacgtgg agcttaacca gtgatagaac tagaaattgg 10440
gttcttcaac agaaaataga aggagaaaca aaagaatcaa actacgctaa attgattgaa 10500
atgaatggag gaggaaccgg ctgtaatcat gaattagaaa tgatcagaca aaagcttcaa 10560
tgtgtagctt caaaactaca ggttctaccc cagaaagcct ctgagagact acagtttgaa 10620
acagcagatg atgaagattt catttgggtt caggaaaata ttgatgaaat tattttacaa 10680
ctacagaaat taactggcca gcaaggtgaa gagcccagct tggtgtcccc aagtacttct 10740
tgtggctcat tgactgaaag actactgaga caaaatgctg agctgacagg gcatatcagt 10800
caactgactg aagagaagaa tgacttaagg aacatggtta tgaagctgga agagcagatc 10860
aggtggtatc gacagacagg agctggtaga gataattctt ccaggttttc attgaatggt 10920
ggtgccaaca ttgaagccat cattgcctct gaaaaagaag tatggaacag agaaaaattg 10980
actctccaga aatctttgaa aagggcagag gctgaagtat acaaactgaa agctgaacta 11040
agaaatgact ctttacttca aactctgagc cctgattctg aacatgtcac tttaaagaga 11100
atttatggta aatacttgag ggcagaaagt tttcgaaagg ctctcattta ccagaagaaa 11160
tacctgctgc tgttactggg tgggttccag gaatgtgaag atgccacctt ggccctgctt 11220
gcccggatgg gggggcagcc agctttcacg gatctagagg tgatcaccaa tcgcccaaag 11280
ggcttcacca ggtttcggtc ggccgtcaga gtatccattg caatttccag aatgaaattt 11340
ttggttcgac ggtggcatcg agtcacaggt tctgtttcca tcaatattaa cagagatggc 11400
tttggactga atcaaggtgc agaaaagact gactcatttt atcattcttc tggtgggctg 11460
gagttatatg gagaaccaag acatactacg tatcgctcaa gatcagatct ggactatatt 11520
aggtcccctt taccatttca gaataggtac ccaggcactc cagctgattt caatcctggt 11580
tctttagcat gttctcagct tcagaattac gatcctgaca gagccctaac agattatatc 11640
actcggctag aggcactgca aagacgactt ggaactatac agtcaggttc aactactcaa 11700
tttcatgctg gcatgagaag ataa 11724
<210> 2
<211> 11724
<212> DNA
<213> Homo sapiens
<400> 2
atggaggacg aggagagaca gaagaagctg gaggccggca aagccaagct tgcccagttt 60
cgacaaagaa aagctcagtc ggatgggcag agtccttcca agaagcagaa aaaaaagaga 120
aaaacgtcaa gcagtaaaca tgatgtgtca gcacaccatg atttgaatat tgatcaatca 180
cagtgtaatg aaatgtacat aaatagttct cagagagtag aatcaactgt gattcctgaa 240
tctacaataa tgagaactct acatagtgga gaaataacca gtcatgagca gggcttctct 300
gtggaactgg aaagtgaaat ttcaaccaca gcagatgact gcagttcaga ggtaaatggt 360
tgcagttttg tgatgagaac aggaaagcct acaaatttat taagggaaga agaatttggt 420
gttgatgatt cttattctga acaaggagca caagacagtc cgactcatct agagatgatg 480
gaaagtgagt tggctgggaa gcagcatgag attgaagagc taaacagaga gctggaagaa 540
atgagggtta cctatgggac tgaaggactg cagcagttac aagaatttga agctgccatt 600
aaacaaagag atggcattat aacccagctc actgctaatt tacaacaagc aagaagagaa 660
aaggatgaga caatgagaga atttttagag ttgacagaac agagtcaaaa attacagatt 720
caatttcagc aattacaggc tagtgaaact ctgagaaaca gcactcatag tagcacagct 780
gcagacttac tacaagccaa acaacagatc ctcactcatc aacagcagct tgaagaacaa 840
gaccacttat tagaagatta tcagaaaaag aaagaagact tcacaatgca aattagtttc 900
ttgcaagaga aaattaaagt atatgaaatg gaacaagata aaaaagtaga aaactcaaat 960
aaagaagaaa tacaggaaaa ggagacaatc attgaagaat taaacacaaa aataatagaa 1020
gaagaaaaga aaactcttga gctaaaggat aaattaacaa ctgctgataa attactagga 1080
gaattacaag aacagattgt gcaaaagaac caagaaataa aaaacatgaa attagagctg 1140
actaattcta agcaaaaaga aagacagtct tctgaagaaa taaaacagtt aatggggaca 1200
gtcgaagaac ttcagaagag aaatcataaa gacagccagt tcgaaactga tatagtacaa 1260
cgaatggaac aagaaacaca aagaaagtta gaacaactcc gggcagagct ggatgagatg 1320
tatgggcagc agatagtgca aatgaaacaa gaattaataa gacaacacat ggcacagatg 1380
gaggaaatga aaacacggca taagggagaa atggagaatg ctttaaggtc atattcaaat 1440
attacagtta atgaagatca gataaagtta atgaatgtgg caataaatga actgaatata 1500
aaattgcaag atactaactc tcaaaaggaa aaactcaagg aagaactagg actaatttta 1560
gaagaaaagt gtgctctaca gagacagctt gaagaccttg ttgaagaatt gagcttttca 1620
agggaacaga ttcagagagc tagacagaca atagctgaac aagaaagtaa acttaatgaa 1680
gcacataagt cccttagtac agtggaagat ttgaaagctg agattgtttc tgcatctgaa 1740
tccagaaagg aactagaatt aaaacatgaa gcagaagtta caaattacaa gataaaactt 1800
gaaatgttag aaaaagaaaa gaatgctgtg ttagacagaa tggctgaatc acaagaagct 1860
gaattagaga ggctgagaac acagcttcta tttagtcacg aagaagagct ttccaaactg 1920
aaggaagatt tagaaattga acatcgaata aatattgaaa aacttaaaga taatttaggc 1980
attcactata aacagcagat agatggttta cagaatgaaa tgagtcaaaa gatagaaacc 2040
atgcagtttg aaaaggacaa tttgataact aagcagaatc aattaatttt ggaaatttca 2100
aagctaaaag atttacagca gtctcttgta aattcaaagt cagaagaaat gactcttcaa 2160
atcaatgaac ttcaaaaaga aattgaaata ctcagacaag aagaaaaaga aaagggtaca 2220
cttgaacaag aagttcaaga attacaactt aaaacagaat tgttagaaaa acagatgaag 2280
gaaaaagaga atgatcttca agaaaaattt gcacaacttg aagcagagaa tagcattctt 2340
aaagatgaaa agaaaaccct tgaagacatg ttgaaaatac atactcctgt tagccaagaa 2400
gaaagattga ttttcttaga ctccattaag tccaaatcca aagactctgt gtgggaaaaa 2460
gaaatagaaa tacttataga ggaaaatgag gacctcaaac aacaatgtat tcagctaaat 2520
gaagagattg aaaagcaaag gaacactttt tcatttgctg aaaaaaactt tgaagttaac 2580
tatcaagagt tacaagagga gtatgcttgc cttctcaaag taaaagatga tttagaagac 2640
agtaaaaata aacaggaatt agagtataaa agtaaactta aagcacttaa tgaagagctt 2700
catttgcaaa gaataaatcc aactacagtg aaaatgaaaa gttctgtctt tgatgaagac 2760
aaaacttttg tagcagaaac attggaaatg ggtgaggttg ttgaaaagga tacaacagaa 2820
ctcatggaaa aacttgaggt aaccaagcga gagaaattag agctgtcaca gagactgtct 2880
gatctttctg aacaattgaa acagaaacat ggtgagatta gttttctaaa tgaagaagtt 2940
aaatctttaa agcaagagaa agaacaagtt tcattgagat gtagagagct agaaatcatt 3000
attaaccaca acagggcaga aaatgtacag tcatgtgata ctcaagtaag ctctttatta 3060
gatggagttg tgaccatgac aagcaggggt gctgaaggat cagtttctaa agtaaataaa 3120
agttttggtg aagaatcaaa aataatggtg gaagataaag tttcttttga aaatatgact 3180
gttggagaag aaagtaagca agaacagttg attttggatc acttaccatc tgtaacaaag 3240
gaatcatcac ttagagcaac tcaaccaagt gaaaatgata aacttcagaa agaactcaat 3300
gtacttaaat cagaacagaa tgatttaagg ctacagatgg aagcccaacg catttgcctc 3360
tctctggttt attcaactca tgtggatcag gttcgtgaat atatggaaaa tgaaaaagat 3420
aaagctcttt gcagtcttaa agaagagctt atttttgctc aagaggaaaa gatcaaggaa 3480
cttcagaaaa tacaccagtt agaactacag actatgaaaa cacaagaaac aggtgatgaa 3540
ggaaagcctt tacatctgct cattggaaaa cttcaaaagg cagtgtctga agaatgttct 3600
tattttttac agactttatg cagtgtcctt ggtgaatatt atactcctgc tttaaaatgt 3660
gaagtaaatg cagaagacaa agagaattct ggtgattaca tttctgaaaa tgaagatcca 3720
gaattacaag attatagata tgaagttcaa gactttcaag aaaatatgca cactcttctc 3780
aacaaagtaa cagaagaata caacaaactc ttggtacttc aaacacgact aagcaagatc 3840
tggggacagc agacagatgg tatgaaactt gaatttggag aagaaaacct tccaaaagag 3900
gaaacagagt ttttatcaat ccattctcag atgaccaatt tggaagacat tgatgtcaat 3960
cataaaagca agttatcttc tctgcaagat cttgaaaaaa ctaaacttga agaacaagtt 4020
caagaattag aaagcctcat atcctctttg cagcaacagt tgaaagaaac tgaacaaaac 4080
tatgaggcag agatccactg tttacagaag aggcttcaag ctgttagtga gtccacggtt 4140
ccgccaagct tacctgttga ttcggtggta attacagaat ctgatgcaca gagaacaatg 4200
taccctggaa gttgtgtgaa aaagaatatt gatggtacaa tagagttttc tggtgaattt 4260
ggagtgaaag aggaaacaaa tatcgttaag ttgcttgaaa aacaatacca agaacaatta 4320
gaagaagaag tagctaaggt tattgtgtca atgagtatag catttgctca acaaactgaa 4380
ctgtctagaa tatctggggg aaaagaaaat actgcatcat caaagcaagc acatgctgtg 4440
tgtcagcaag aacaacatta ttttaatgaa atgaaattat cacaggatca aattggtttt 4500
cagacttttg agacagtgga tgtgaaattt aaagaagaat ttaaaccact tagtaaagag 4560
ttaggagaac atggaaagga aattttatta tcaaatagtg atccccatga tataccagaa 4620
tcaaaggact gtgtgctgac tatttcagaa gaaatgttct ccaaagataa aacatttata 4680
gttagacagt ctattcatga tgagatttca gtgtcaagca tggatgcttc tagacaacta 4740
atgttgaatg aagaacagtt ggaagatatg agacaggaac ttgtacgaca ataccaagaa 4800
catcaacagg caacggaatt gttaaggcaa gcacatatgc ggcaaatgga gagacagcga 4860
gaagaccagg aacagctaca agaagagatt aagagactta atagacaatt agcccagaga 4920
tcctccatag ataatgaaaa cctggtttca gagagagaga gggtgctttt agaggagctg 4980
gaagcactaa agcagctgtc tttagctgga agagagaagc tgtgttgtga gctgcgcaac 5040
agcagtacgc aaacacagaa tggaaatgaa aaccaaggag aagttgaaga acaaacattt 5100
aaagaaaagg aattagacag aaaacctgaa gatgtgcctc ctgagatttt gtctaatgaa 5160
aggtatgcac tccagaaagc taataataga cttttgaaga tcctcttaga agttgtaaag 5220
acaacagcag ctgttgaaga aacaattggt cgccatgtcc ttgggattct agatagatct 5280
agtaaaagcc agtcatctgc cagcctaatt tggaggtcag aagcagaggc atctgtaaag 5340
tcatgtgtcc atgaggaaca tacaagagtt acagatgaat ccattccctc ttattctgga 5400
agtgatatgc caagaaatga cattaacatg tggtcaaaag taactgagga aggaacagag 5460
ctgtcacaac gacttgtgag gagtggtttt gctggaactg aaatagaccc tgaaaatgaa 5520
gaacttatgc tgaacattag ctctcgacta caagcagcag ttgaaaaact cctagaagcc 5580
ataagtgaaa ctagcagtca gcttgaacat gcgaaagtga cacagacaga gttgatgcgt 5640
gagtcattta gacagaaaca agaagcaaca gagtccctta agtgccaaga ggaacttcga 5700
gagcgccttc atgaggagtc cagggccaga gaacagctag ctgtggagct cagtaaggct 5760
gagggcgtca ttgatggcta tgcagatgaa aaaactcttt ttgaaaggca aattcaggaa 5820
aaaactgata taatagatcg tcttgagcag gagttgttat gtgcaagtaa caggttgcaa 5880
gaattggagg cagagcaaca gcagatccaa gaagaaagag aattactgtc cagacaaaag 5940
gaagctatga aagcagaggc aggcccagtt gaacaacaat tactacagga gacagaaaaa 6000
ttaatgaagg aaaaactaga agtacaatgt caagctgaaa aagtacgtga tgaccttcaa 6060
aaacaagtga aagctctaga aatagatgtg gaagaacaag tcagtaggtt tatagagctg 6120
gaacaagaaa aaaatactga actaatggat ttaagacagc aaaaccaagc attggaaaag 6180
cagttagaaa aaatgagaaa atttttagat gagcaagcca ttgacagaga acatgagaga 6240
gatgtattcc aacaggaaat acagaaacta gaacagcaac ttaaggttgt tcctcgattc 6300
cagcctatca gtgaacatca aactagagag gttgaacagt tagcaaatca tctgaaagaa 6360
aaaacagaca aatgcagtga gcttttgctc tctaaagagc agcttgaaag ggatatacaa 6420
gaaaggaatg aagaaataga gaaactggag ttcagagtaa gagaactgga gcaggcgctt 6480
cttgtgagtg cagatacttt tcaaaaggta gaggaccgaa aacactttgg agctgtagaa 6540
gctaaaccag aattgtccct agaagtacaa ttgcaggctg aacgagatgc catagacaga 6600
aaggaaaaag agattacaaa cttagaagag caattagaac agtttagaga agaactggaa 6660
aataagaatg aagaagttca acaattacat atgcaattag aaatacagaa aaaggaatct 6720
actacccgcc tacaagaact tgaacaggaa aacaaattat ttaaggatga catggagaaa 6780
ctgggacttg ccataaagga atctgatgcc atgtctactc aagaccaaca tgtgctattt 6840
gggaaatttg ctcaaataat acaggaaaaa gaggtagaaa ttgaccaatt aaatgaacaa 6900
gttacgaaac tccagcagca acttaaaatt acaacagata acaaggttat tgaagaaaaa 6960
aatgaactga taagggatct tgaaacccaa atagaatgtt tgatgagtga tcaagaatgt 7020
gtgaagagaa atagagaaga agaaatagag cagctcaatg aagtgattga aaaacttcaa 7080
caggaattgg caaatattgg acagaagaca tcaatgaatg ctcattccct ctcagaagaa 7140
gcagacagtt taaaacatca attggatgtg gttatagctg aaaagctggc cttggaacag 7200
caagtagaaa ccgctaatga agaaatgacc ttcatgaaaa atgtacttaa agaaaccaat 7260
tttaaaatga atcagctaac acaggaatta ttcagcttaa agagagaacg tgaaagtgtg 7320
gaaaagattc aaagcatacc agagaatagt gttaacgtgg ctatagatca tctgagcaaa 7380
gacaaacctg aactagaagt agtccttaca gaggatgctc ttaaatccct agaaaatcag 7440
acatacttca aatcttttga agaaaatggc aaaggttcca taattaattt ggaaacaagg 7500
ttgctacaac ttgagagcac tgttagtgca aaggacttag aacttaccca gtgttataaa 7560
caaataaaag acatgcaaga acaaggccag tttgaaacag aaatgcttca aaagaagatt 7620
gtaaacctac agaaaatagt tgaagaaaaa gtggctgctg ctcttgtcag tcaaatccaa 7680
cttgaggcag ttcaggaata tgcaaaattc tgtcaagata atcaaacaat ttcatcagaa 7740
cctgaaagaa caaatattca gaatttaaat caactaagag aagatgagtt ggggtcagat 7800
atatcagcat taaccttgag aatatcagaa ttagaaagcc aggttgttga aatgcatact 7860
agtttgattt tagaaaaaga acaagtagaa attgcagaaa aaaatgtttt agaaaaagaa 7920
aagaagctgc tagaactaca gaagctattg gagggcaatg agaaaaaaca gagagagaaa 7980
gaaaagaaaa gaagccctca agatgttgaa gttctcaaga caactactga gctatttcat 8040
agcaatgaag aaagtggatt ttttaatgaa ctcgaggctc ttagagctga atcagtggct 8100
accaaagcag aacttgccag ttataaagaa aaggctgaaa aacttcaaga agagcttttg 8160
gtaaaagaaa caaatatgac atctcttcag aaagacttaa gccaagttag ggatcacctc 8220
gcagaggcaa aagagaaatt gtccatttta gaaaaagaag atgagactga ggtacaagaa 8280
agcaaaaagg cctgcatgtt tgagccactt cctataaaac tgagtaagag cattgcatcc 8340
cagacagatg ggactctgaa gatcagtagc agcaatcaga ctccacaaat tcttgttaaa 8400
aatgcaggaa tacaaattaa tttacagagt gaatgttcct cagaagaagt tactgaaata 8460
atcagtcagt ttactgaaaa aattgagaag atgcaagaac tacatgctgc tgaaattttg 8520
gacatggaat ccagacatat ttcagaaact gaaaccttaa agagggaaca ctatgttgcc 8580
gttcagttac tgaaagagga atgtggtacc ttgaaggcag tgatacagtg tctgagaagt 8640
aaagagggat cctcaattcc tgagctagca cattctgatg cttaccagac tagagaaata 8700
tgctccagtg attctggatc agactggggt cagggaattt atcttacaca cagtcaggga 8760
tttgacatag catcagaagg ccgaggagaa gaaagtgaaa gtgcaacaga ttcctttcca 8820
aagaaaataa agggattact gagagctgtc cataatgaag gcatgcaggt gctttctctc 8880
actgagtctc cctatagtga tggagaggac cattctattc agcaggtttc agaaccttgg 8940
ctagaagaga gaaaagctta catcaataca atctcatctc taaaggattt aattacaaag 9000
atgcaactgc aaagagaagc cgaggtttat gatagttctc aatctcatga gagcttctca 9060
gactggcgag gtgaactact gcttgccctt caacaagttt tcttagaaga gcgtagtgtt 9120
ttactagcag catttcggac ggagctgaca gctctaggta ctacagatgc agttggttta 9180
ctaaactgtt tggaacagag aatacaagaa cagggtgttg aatatcaagc agctatggaa 9240
tgcctccaga aagcagatag aaggagtttg ttatctgaaa ttcaggcact gcatgcacaa 9300
atgaatggta ggaaaattac tctgaaaaga gaacaagaga gtgagaaacc aagccaagaa 9360
ctcttggaat ataatataca gcagaagcag tctcaaatgc tggagatgca agtggagctc 9420
agcagtatga aagacagagc aacggaactg caggagcagc tgagttctga gaaaatggtg 9480
gttgctgaac tgaagagtga gcttgcacaa actaaattgg aactagaaac aacactcaag 9540
gcacagcata aacacctaaa agaattggag gctttcaggt tggaagttaa agataagaca 9600
gatgaagtac atttgcttaa tgacacatta gcaagtgaac agaaaaaatc aagagagctc 9660
cagtgggctt tggagaaaga gaaagccaag ttgggacgca gtgaagaacg ggataaagaa 9720
gaacttgagg atctgaagtt ttcacttgag agtcagaaac aaaggaatct tcagctaaat 9780
ctacttttgg aacaacagaa acaactactg aacgaatccc agcaaaaaat agaatcacag 9840
agaatgctat atgatgccca gttgtcagaa gaacaaggtc gaaacttaga gcttcaggta 9900
cttcttgaat ctgagaaagt tcgaattcgg gaaatgagta gtaccctaga tagggagcgg 9960
gaattgcacg cacagctgca gagcagtgat ggtactggac agtctcggcc acccttgccc 10020
tcagaggacc tactgaaaga gctgcagaaa cagctagagg aaaaacacag tcgcatagta 10080
gaattgttaa atgagactga aaaatataaa ctggattctt tgcaaacacg acagcaaatg 10140
gaaaaagata ggcaggttca caggaaaaca ctgcagacag aacaggaggc caacactgag 10200
ggacagaaaa aaatgcatga gctccagtcc aaagtggaag atcttcagcg ccagctggaa 10260
gagaaaagac aacaagttta taagttagac cttgaaggac agcgactaca aggaatcatg 10320
caggaattcc agaagcaaga actagaacga gaagaaaaac gagaaagtag aagaattctg 10380
tatcagaacc ttaatgagcc aaccacgtgg agcttaacca gtgatagaac tagaaattgg 10440
gttcttcaac agaaaataga aggagaaaca aaagaatcaa actacgctaa attgattgaa 10500
atgaatggag gaggaaccgg ctgtaatcat gaattagaaa tgatcagaca aaagcttcaa 10560
tgtgtagctt caaaactaca ggttctaccc cagaaagcct ctgagagact acagtttgaa 10620
acagcagatg atgaagattt catttgggtt caggaaaata ttgatgaaat tattttacaa 10680
ctacagaaat taactggcca gcaaggtgaa gagcccagct tggtgtcccc aagtacttct 10740
tgtggctcat tgactgaaag actactgaga caaaatgctg agctgacagg gcatatcagt 10800
caactgactg aagagaagaa tgacttaagg aacatggtta tgaagctgga agagcagatc 10860
aggtggtatc gacagacagg agctggtaga gataattctt ccaggttttc attgaatggt 10920
ggtgccaaca ttgaagccat cattgcctct gaaaaagaag tatggaacag agaaaaattg 10980
actctccaga aatctttgaa aagggcagag gctgaagtat acaaactgaa agctgaacta 11040
agaaatgact ctttacttca aactctgagc cctgattctg aacatgtcac tttaaagaga 11100
atttatggta aatacttgag ggcagaaagt tttcgaaagg ctctcattta ccagaagaaa 11160
tacctgctgc tgttactggg tgggttccag gaatgtgaag atgccacctt ggccctgctt 11220
gcccggatgg gggggcagcc agctttcacg gatctagagg tgatcaccaa tcgcccaaag 11280
ggcttcacca ggtttcggtc ggccgtcaga gtatccattg caatttccag aatgaaattt 11340
ttggttcgac ggtggcatcg agtcacaggt tctgtttcca tcaatattaa cagagatggc 11400
tttggactga atcaaggtgc agaaaagact gactcatttt atcattcttc tggtgggctg 11460
gagttatatg gagaaccaag acatactacg tatcgctcaa gatcagatct ggactatatt 11520
aggtcccctt taccatttca gaataggtac ccaggcactc cagctgattt caatcctggt 11580
tctttagcat gttctcagct tcagaattac gatcctgaca gagccctaac agattatatc 11640
actcggctag aggcactgca aagacgactt ggaactatac agtcaggttc aactactcaa 11700
tttcatgctg gcatgagaag ataa 11724
<210> 3
<211> 18
<212> DNA
<213> Homo sapiens
<400> 3
gtagatatcc aaaatgag 18
<210> 4
<211> 20
<212> DNA
<213> Homo sapiens
<400> 4
aatggttaca atgtcaacag 20

Claims (9)

1.一种AKAP9的基因突变，其特征在于，所述AKAP9的基因突变与正常AKAP9基因相比具有c.6406C>G突变。
2.一种核酸，其特征在于，与正常AKAP9基因相比，所述核酸具有c.6406C>G突变。
3.一种多肽，其特征在于，与正常AKAP9基因表达的多肽的氨基酸序列相比，所述多肽的氨基酸序列具有p.Q2136E突变。
4.检测权利要求1所述的基因突变或权利要求2所述的核酸或权利要求3所述的多肽的试剂在制备试剂盒或者设备中的用途，所述试剂盒或者设备用于诊断冠心病；
优选的，所述冠心病为早发冠心病；
优选的，所述试剂包括特异性针对所述基因突变、所述核酸和所述多肽的至少之一的抗体、探针、引物以及质谱检测试剂的至少之一。
5.生物模型在筛选药物中的用途，其特征在于，所述生物模型选自下列至少之一：
(1)权利要求1所述的基因突变；
(2)权利要求2所述的核酸；
(3)权利要求3所述的多肽；
优选地，所述生物模型为细胞模型或者动物模型。
6.特异性改变权利要求1所述的基因突变或者权利要求2所述的核酸的试剂在制备药物中的用途，所述药物用于治疗冠心病；
优选地，所述冠心病为早发冠心病。
7.一种用于治疗冠心病的药物，其特征在于，所述药物含有：特异性改变权利要求1所述的基因突变或者权利要求2所述的核酸的试剂；
优选地，所述冠心病为早发冠心病。
8.一种检测冠心病的试剂盒，其特征在于，所述试剂盒中包括检测权利要求1所述的基因突变的试剂，和/或检测权利要求2所述的核酸的试剂，和/或检测权利要求3所述的多肽的试剂。
9.一种筛选与早发冠心病相关突变位点的筛选方法，其特征在于，所述方法包括
以下步骤：
(1)提取早发冠心病患者和正常对照的外周血样本的DNA；
(2)将DNA进行超声片段化，随机打断成长度为180-280bp的片段，经末端修复和加A尾后在片段两端分别连接上接头制备DNA文库；
(3)采用Agilent的液相芯片捕获系统，对人的全外显子区域DNA进行高效富集，然后在Illumina平台上进行高通量双端测序，每端读长为150bp；
(4)外显子测序后，进行数据质量评估，对测序错误率、数据量和比对率进行统计，并将有效测序数据以GRCh37/hg19为参考序列或参考基因组通过Burrows-Wheeler Aligner进行序列比对，使用SAMtools对比对结果进行排序，再用Sambamba标记重复reads，最后利用重复标记后的比对结果进行覆盖度、深度的统计；
(5)在上述比对结果的基础上利用GATK4识别SNP，InDel以及CNV，利用ANNOVAR软件对上述位点进行注释，其中包括dbSNP数据库、千人基因组计划和其他已有的数据库的注释信息，注释内容涵盖变异的位置信息，类型，保守型预测，主要步骤包括碱基质量重矫正，局部重比对以及基于单倍型的基因分型；生成的变异位点文件进一步通过ANNOVAR软件进行基因组信息以及功能注释；
(6)对变异检测的结果，通过基于人群频率、变异的功能有害性预测、物种保守性分析以及所在基因的组织表达水平进行筛选；最后对候选的变异位点结合临床表型进行遗传解读，包括遗传位点数据库检索，家系疾病共分离分析，以及ACMG致病性评级；最终筛选出早发冠心病易感性SNP位点：AKAP9_c.6406C>G_p.Q2136E。
CN202210238216.6A 2022-03-11 2022-03-11 早发冠心病相关的snp位点及其应用 Pending CN114457087A (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070082347A1 (en) * 2005-06-08 2007-04-12 Myriad Genetics, Incorporated Gene variants and use thereof
US20160281166A1 (en) * 2015-03-23 2016-09-29 Parabase Genomics, Inc. Methods and systems for screening diseases in subjects
CN109652531A (zh) * 2019-01-11 2019-04-19 中国人民解放军总医院 一种用于检测遗传性心肌病/心律失常的致病/易感基因的探针组
CN109750099A (zh) * 2019-01-30 2019-05-14 中国人民解放军总医院 一种用于检测心源性猝死的致病/易感基因的探针组

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070082347A1 (en) * 2005-06-08 2007-04-12 Myriad Genetics, Incorporated Gene variants and use thereof
US20160281166A1 (en) * 2015-03-23 2016-09-29 Parabase Genomics, Inc. Methods and systems for screening diseases in subjects
CN109652531A (zh) * 2019-01-11 2019-04-19 中国人民解放军总医院 一种用于检测遗传性心肌病/心律失常的致病/易感基因的探针组
CN109750099A (zh) * 2019-01-30 2019-05-14 中国人民解放军总医院 一种用于检测心源性猝死的致病/易感基因的探针组

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