CN105492631B - 一种与特发性无精子症相关的遗传标记 - Google Patents
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Abstract
本发明公开了一种与特发性无精子症相关的遗传标记，其位于AR基因的编码区，其核苷酸序列如SEQ ID NO:1所示，其中，所述序列的突变位点选自c.868 T>C、c.1484G>A、c.1888 C>T、c.569 C>T、c.616 A>G和c.1149 C>T中的一个或多个。通过大规模测序在特发性无精子症病人中筛选出了AR基因的6个新的突变位点，构建AR基因野生型及突变体表达质粒转染到细胞内进行研究，通过实验发现其功能有明显差异，表明AR基因的上述突变可能导致特发性无精子症的发生，从而能够通过上述突变来实现对男性不育症（尤其是特发性无精子症）的诊断检测。
Description
技术领域
本发明涉及男性不育症检测领域，尤其涉及一种与特发性无精子症相关的遗传标记。
背景技术
全球约有10％～15％的育龄夫妇面临不能生育的问题，其中一半是由于男性不育。原发性无精子症是造成男性不育的一个非常重要的原因，约影响1％的成年男性。男性不育发病因素具有复杂性和多样性的特点，包括疾病、营养不良、内分泌紊乱、基因缺陷和环境因素等，其具体发病机制尚不明确，但从家族病例报道和小鼠模型的研究成果中可以推断遗传因素起了很大作用。近年来，随着现代分子生物学技术的发展，人们已发现近200个基因与男性不育症的发生密切相关；采用基因敲除技术，发现近400个基因与小鼠精子发生密切相关，这些基因的突变、缺失或表达异常，可能是男性不育症发生的重要原因。对这些突变基因的研究将有助于男性不育症的诊断，也有助于将来在体外受精过程中防止将遗传缺陷带给下一代。
雄激素受体(AR，NCBI Gene ID:367)是一种重要的类固醇激素受体，在男性性分化和维持正常精子发生过程中发挥关键作用。AR属于核转录因子调控的类固醇激素作用的家族。AR有4个蛋白结构，包括N末端反式激活结构域(NTD)，DNA结合域(DBD)，铰链区(HR)和羧基配体结合域(LBD)。它能结合雄激素，包括睾酮(T)和5α-二氢睾酮(DHT)的配体结合域，从而介导核易位和AR的转录调节功能。
在过去的几年中，确定了一些AR突变或多态性，可以导致或与遗传疾病相关，如完全或部分雄激素不敏感综合征(CAIS和PAIS)。然而，还需要深入地研究以揭示AR突变与特发性无精子症(IA)的关系，为特发性无精子症的诊断提供依据和指导。
发明内容
为了确定IA患者AR基因突变的情况，我们将776例IA患者和709例正常生育男性的AR基因外显子测序，首次新发现5个错义突变和1个同义突变与IA的发生相关。
基于本发明的发现，本发明提供一种与特发性无精子症相关的遗传标记，其位于AR基因的编码区，其核苷酸序列如SEQ ID NO:1所示，其中，上述序列的突变位点选自c.868T>C、c.1484G>A、c.1888C>T、c.569C>T、c.616A>G和c.1149C>T中的一个或多个。
前5个突变位点是错义突变，第6个突变位点是同义突变。
上述突变分别是在如SEQ ID NO:1所示的AR基因序列的编码区的第868位、1484位、1888位、569位、616位、1279位和1149位。
前5个突变位点依次分别对应的氨基酸变化情况是p.C290R、p.S495N、p.R630W、p.T190I和p.S206G，即第290位氨基酸由C变为R、第495位氨基酸由S变为N、第630位氨基酸由R变为W、第190位氨基酸由T变为I、第206位氨基酸由S变为G。第6个突变位点没有对应的氨基酸变化。
作为本发明的优选方案，上述序列的突变位点选自c.868T>C、c.1484G>A和c.1888C>T中的一个或多个。
作为本发明的优选方案，上述序列的突变位点选自c.1888C>T。
通过大规模测序在特发性无精子症病人中筛选出了AR基因的6个新的突变位点，构建AR基因野生型及突变体表达质粒转染到细胞内进行研究，通过实验发现其功能有明显差异，表明AR基因的上述突变可能导致特发性无精子症的发生，从而能够通过上述突变来实现对男性不育症(尤其是特发性无精子症)的诊断检测。
附图说明
图1为无精子症患者AR基因的3个错义突变位点测序图谱；
图2为无精子症患者AR基因的3个错义突变位点影响的进化保守性的氨基酸；
图3为在AR基因上发现的3个IA病人特异的错义突变的位置；
图4为3个AR突变体对下游靶基因MMTV表达的影响，NC表示阴性对照，WT表示野生型。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。
1实验内容
1.1标本的收集
本申请人从2007年6月到2011年10月共收集1880例无精子症病人，其中特发性无精子症病人为776例，我们的筛选标准为：1)随机检查三次精液中无精子；2)生殖系统或盆腔无阻塞、炎症和损伤；3)无核型异常和Y染色体微缺失，另将709例正常可育男性(至少育有一个孩子且未行IVF、ICSI、IMSI等人类辅助生殖技术)作为对照进行研究。每例受试者均认真签署知情同意书，本次研究通过医院伦理委员会的审查批准。
1.2外显子测序
抽取外周血提取基因组DNA，用枸橼酸钠抗凝管收集研究对象的外周血，迅速置于-80℃冰箱中备用，用QIAamp DNA Blood Mini Kit试剂盒提取外周血DNA。
取出5微克基因组DNA送华大基因研究中心(深圳)进行外显子捕获、测序，在特发性无精子症患者中筛选出AR基因中的9个突变位点中，其中7个错义突变和2个同义突变，与dbSNP135数据库、千人基因组数据库和ExAC数据库中的数据相比对，有5种错义突变和1个同义突变是我们首次发现的新突变。
1.3验证错义突变
以提取的外周血基因组DNA为模板，使用设计合成的3对引物对仅在特发性无精子症患者(W320，W691，W530)AR基因存在的3个错义突变位点(c.868T>C、c.1484G>A和c.1888C>T)进行特异性扩增，AR序列从NCBI数据库中查得(NCBI Gene ID:367)。引物由Invitrogen公司合成，所合成的3对引物对见表1。
表1 AR突变位点验证引物
PCR扩增条件为：98℃预变性2min，然后以98℃10s、60℃30s、72℃45s进行35个循环，最后72℃延伸5min。
DNA电泳：取3μl的PCR产物在1％琼脂糖凝胶孔中，140V电泳，15min，紫外凝胶成像系统拍照观察电泳图，确保单一条带，其余PCR产物送上海英潍捷基公司测序，引物对Primer1、Primer2和Primer3扩增到的PCR产物片段序列分别如序列表中SEQ ID NO:8、SEQID NO:9和SEQ ID NO:10所示。
1.4 AR突变体表达质粒的构建
以pcDNA3.1-AR(Mou L等人,Identification of Ube2b as a novel tARget ofandrogen receptor in mouse sertoli cells.BiolReprod.2013 Aug 15；89(2):32.)为模板，使用设计合成的3对点突变引物，构建AR的3种突变体表达质粒。引物由Invitrogen公司合成，所合成的3对点突变引物见表2。
表2 AR 3对定点突变引物
1.5双荧光素酶报告基因实验
1.5.1质粒准备
野生型AR表达载体：pcDNA3.1-AR WT(WT组)
突变型AR表达载体：pcDNA3.1-AR C290R(C290R组)
pcDNA3.1-AR S495N(S495N组)
pcDNA3.1-AR R630W(R630W组)
1.5.2 HeLa细胞培养
将HeLa细胞用含10％胎牛血清DMEM培养基在37℃、5％CO2和95％湿度条件下培养。贴壁细胞在长满瓶底时，用0.25％胰蛋白酶消化后按比例进行传代培养。
1.5.3 HeLa细胞瞬时转染
将HeLa细胞接种于24孔板，待细胞贴壁后进行转染，方法参考转染试剂LipofectamineTM 2000说明书。转染6h后，用移液枪吸弃每孔培养液，每孔再加入新的1640培养基500μl，减少LipofectamineTM 2000对细胞的毒性。
1.5.4双荧光素酶报告系统检测转录因子活性
1)转染24h后收集细胞，用移液枪吸弃每孔培养液，每孔加入PBS 1ml洗涤2次；
2)将5×Passive Lysis Buffer稀释成1×Passive Lysis Buffer，用移液枪向每孔加入100μl Passive Lysis Buffer；
3)室温下在摇床上裂解细胞15min，用微量移液枪分别吸取每孔细胞裂解液5μl至一个干净的1.5mL透明EP中；
4)在避光环境中依次向每个EP管中加入19μl Luciferase Assay Ⅱ Buffer后用单管光度计检测萤火虫荧光照度；
5)在避光环境中依次再向每个EP管中加入19μl STOP Buffer后用单管光度计检测海肾荧光照度，以萤火虫荧光来校正海肾荧光减少实验误差。测定报告基因的相对活性。
1.6统计学分析
所用统计学方法来自于SPSS17.0软件，每组数据以均数±标准差(x±s)表示，组间均值的比较采用独立样本t检验，P<0.05为有统计学意义。
2实验结果
2.1特发性无精子症患者AR基因点突变的鉴定
对776例特发性无精子症患者和709例正常可育男性的AR基因的外显子测序结果分析，如表3所示：AR基因的9个突变均为纯合突变，前7个为错义突变，最后2个为同义突变。与dbSNP135数据库、千人基因组数据库和ExAC数据库中的数据相比对，有5个错义突变(c.868T>C，c.1484G>A，c.1888C>T，c.569C>T，c.616A>G)和1个同义突变(c.1149C>T)是我们首次发现的新突变。在709例正常可育男性中有3个错义突变(c.868T>C，c.1484G>A，c.1888C>T)和1个同义突变(c.1149C>T)是不存在的。我们进一步用PCR测序验证这3个错义突变，结果如图1所示。
在不同物种中AR氨基酸序列的同源性比较显示R630W的突变影响了一个高度保守的氨基酸(图2)，图2是采用MegAlign(Demonstration System DNASTAR,Inc.)软件对不同蛋白进行比对得到的，其中AR蛋白的编号如下：人(human)(NP_000035.2)，黑猩猩(chimpanzee)(XP_009437511.1)，恒河猴(rhesus)(NP_001028083.1)，牛(cow)(NP_001231056.1)，大鼠(rat)(NP_036634.1)，小鼠(mouse)(NP_038504.1)和鸡(chicken)(NP_001035179.1)。图2中方框表示突变的位置，*表示保守序列。
基于保守型分析，SIFT和Polyphen 2.0软件分析的结果显示，R630W的突变可能影响蛋白的功能(表4)。这3个错义突变的位置如图3所示。
表3 AR点突变在特发性无精子症组和正常组中的情况
注：病人编号以W开头，编号1-7为错义突变，编号8、9为同义突变。
表4 SIFT和PolyPhen 2.0软件预测错义突变对蛋白功能的影响
注：SIFT得分≤0.05：破坏，≥0.05：容忍；
PolyPhen-2:很可能破坏(概率得分>0.85)，可能破坏(概率得分>0.15)，良性(保持)。
2.2 AR突变体功能的变化
为了评估我们发现的病人特有的AR错义突变是否影响了其调节功能，我们采用双荧光素酶报告基因实验，将MMTV-LUC(Mou L等人,Identification of Ube2b as a noveltARget of androgen receptor in mouse sertoli cells.BiolReprod.2013 Aug 15；89(2):32)与AR野生型及3种突变体质粒分别共转染到HeLa细胞中，由于AR属于配体依赖性转录因子，因此我们转染后用雄激素进行处理，将检测到的萤火虫荧光值/海参荧光值来表示启动子的活性。与野生型AR一致，AR C290R和S495N突变体也可以激活MMTV启动子的活性，仅AR R630W突变体与野生型有显著差异，不能激活MMTV启动子的活性(见图4)。这些结果表明，R630W变体抑制AR转录调控活性。
2.3临床信息及激素水平检测
带有3个错义突变(c.868T>C，c.1484G>A，c.1888C>T)的患者进一步进行了阴囊超声检查，结果显示阴囊睾丸小，具有均匀的回声特性和广泛的低回声；没有观察到固体或囊性病变。表5总结了这三例患者的临床和激素水平数据。这些患者都没有男性不育症的家族史。所有AR突变患者均不存在CAIS或PAIS的个人或家族史。
表5.带有3个错义突变的患者的临床信息及激素水平检测
3讨论
越来越多的证据表明，AR是调节雄激素应答基因表达的配体依赖性转录因子。雄激素和AR对男性生精功能和生育能力至关重要。
虽然已经报道AR基因有超过700个突变和多态性，只有5突变位于外显子1，并在无精子症患者有不同程度生精障碍或MAIS。所有这些患者的外生殖器正常，复杂的临床表现和我们的报告都证实了这个方面。与此同时，雄性AR基因敲除小鼠表现出雌性个体的典型外观，这类似于一个人雄激素不敏感综合征或睾丸女性化变异小鼠。
在本发明的研究中，我们测序了776个IA患者AR的编码序列。R630W突变定位于AR的DNA结合结构域，在776例患者中的发现了一个患者有这个突变，但是不存在于709个生育男性以及报告在公共数据库中的其他个体。使用PolyPhen-2和Sift软件分析R630W突变，预测有对蛋白质结构会产生影响。此外，AR的氨基酸序列的局部比对分析表明，受影响的精氨酸残基在多个物种高度保守，包括鸡。这个突变附件的氨基酸都很保守，提示突变可能对AR的功能有较大的影响。
当我们试图评估AR对不育患者的致病作用时，一个关键的问题要解决的是突变的AR是否影响其转录调节功能。正如预期，我们发现R630W突变影响了AR对MMTV启动子的转录调节功能。
总之，我们通过大规模并行测序技术确定了AR基因的七个错义突变和两个同义突变。通过功能试验表明，R630W突变抑制AR的正常转录调节功能。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。
Claims (1)
1.一种与特发性无精子症相关的遗传标记的突变位点在制备用于检测特发性无精子症的试剂盒中的用途，其特征在于，所述遗传标记位于AR基因的编码区，其核苷酸序列如SEQ ID NO:1所示，其中，所述序列的突变位点选自c.1888C>T。
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| The Androgen receptor gene mutations database World Wide Web Server;McGill;《The Androgen receptor gene mutations database World Wide Web Server》;20140930;http://www.androgendb.mcgill.ca,网页及网页上提供的Database的第1-45页 * |
| 雄激素不敏感综合征;迟铖 等;《中国实用内科杂志》;20061031;第26卷(第20期);1574-1577 * |
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