CN105176976A - 一种检测与非梗阻性无精子症相关的遗传标记的引物对 - Google Patents
一种检测与非梗阻性无精子症相关的遗传标记的引物对 Download PDFInfo
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Abstract
本发明公开了一种检测与非梗阻性无精子症相关的遗传标记的引物对，所述遗传标记的核苷酸序列是DAX-1基因的序列，所述序列的突变位点选自c.152G>A、c.312C>G、c.725C>T、c.766G>C、c.1153G>T、c.1279A>G和c.162G>A中的一个或多个；所述引物对选自primer1、primer2、primer3或primer4引物对。通过大规模测序在非梗阻性无精子症病人中筛选出了DAX-1基因7个新的突变位点，研究表明DAX-1的上述突变可能导致非梗阻性无精子症的发生，从而能够通过使用本发明的引物对检测该突变来实现对男性不育症(尤其是非梗阻性无精子症)的诊断检测。
Description
本申请是申请日为2014年9月2日、申请号为201410444435.5、名称为一种与非梗阻性无精子症相关的遗传标记的申请的分案申请。
技术领域
本申请涉及男性不育症检测领域，尤其涉及非梗阻性无精子症检测领域。
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。大多数基因表达异常可使青春期发育受损，随后由于下丘脑-垂体对性腺或其基因有重要促进作用的因子缺乏，最终引起男性不育。剂量敏感的性别反转先天性肾上腺发育不良基因1(Dosage-sensitivesexreversal,adrenalhypoplasiacriticalregion,onchromosomeXgene1,DAX-1)即属于下丘脑-垂体-性腺轴上的基因，其编码的蛋白是核受体家族的成员。DAX-1作为一种转录因子对垂体促性腺细胞和肾上腺皮质的发育起非常重要的作用。
人类DAX-1基因位于X染色体p21，于1994年克隆成功。该基因编码的蛋白由470个氨基酸组成，是核受体家族中的一员，主要在下丘脑、垂体、肾上腺以及性腺中表达，对垂体促性腺细胞和肾上腺皮质的发育起非常重要的作用。越来越多的研究表明先天性肾上腺发育不良(Adrenalhypoplasiacongenital,AHC)和低促性腺激素性性功能不全(Hypogonadotropichypogonadism,HH)的发病与DAX-1突变有关。由于DAX-1基因位于Xp上的DSS区，当该区双拷贝时常伴有46，XY男性的性反转(女性化)，所以起初DAX-1被认为是卵巢决定基因。但是，随后的条件敲除研究结果表明它在调节精子发生中起重要作用，敲除后的雄性小鼠表现为性腺机能减退、睾丸发育异常、生精障碍等。Holter等人的研究表明，雄激素受体(Androgenreceptor,AR)是DAX-1的一个新的作用靶点，DAX-1通过与AR直接相互作用抑制AR的转录活性。
发明内容
本发明提供一种与非梗阻性无精子症相关的遗传标记。
本发明提供一种与非梗阻性无精子症相关的遗传标记，其核苷酸序列是DAX-1基因的序列，所述序列的突变位点选自c.152G>A、c.312C>G、c.725C>T、c.766G>C、c.1153G>T、c.1279A>G和c.162G>A中的一个或多个。
前6个突变是错义突变，第7个突变是同义突变。
上述突变分别是在DAX-1基因序列的第152位点、312位点、725位点、766位点、1153位点、1279位点和162位点。
一种与非梗阻性无精子症相关的遗传标记，其核苷酸序列是DAX-1基因的序列，所述序列的突变位点选自c.152G>A、c.312C>G、c.725C>T、c.766G>C、c.1153G>T和c.1279A>G中的一个或多个。
所述突变位点是c.1153G>T。
检测所述的遗传标记的引物对，选自primer1、primer2、primer3或primer4引物对，其中，所述primer1引物对的上游引物的序列如SEQIDNO:1所示，其下游引物的序列如SEQIDNO:2所示；所述primer2引物对的上游引物的序列如SEQIDNO:3所示，其下游引物的序列如SEQIDNO:4所示；所述primer3引物对的上游引物的序列如SEQIDNO:5所示，其下游引物的序列如SEQIDNO:6所示；所述primer4引物对的上游引物的序列如SEQIDNO:7所示，其下游引物的序列如SEQIDNO:8所示。
一种非梗阻性无精子症的检测试剂盒，能够特异性识别所述的遗传标记。该遗传标志尤其是c.1153G>T突变。
一种所述的遗传标记在制备非梗阻性无精子症检测试剂盒上的应用。
本发明的有益效果是：通过大规模测序在非梗阻性无精子症病人中筛选出了DAX-1基因7个新的突变位点，构建DAX-1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DAX-1的上述突变可能导致非梗阻性无精子症的发生，从而能够通过该突变来实现对男性不育症(尤其是非梗阻性无精子症)的诊断检测。
附图说明
图1是无精子症患者DAX-1基因的6个错义突变位点测序图谱；
图2是免疫共沉淀检测DAX-1野生型及突变体与AR的结合的结果图；
图3是DAX-1突变体与AR相互作用后对AR下游靶基因MMTV表达的影响的结果图。
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。
1.2实验内容
1.2.1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，其中非梗阻性无精子症病人为776例，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706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。
1.2.2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QIAampDNABloodMiniKit试剂盒提取外周血DNA。
取出5ug基因组DNA送华大基因研究中心(深圳)进行外显子捕获、测序，在非梗阻性无精子症患者中筛选出DAX-1基因中的7个新的突变位点，其中6个错义突变和1个同义突变，与dbSNP135数据库和千人基因组数据库中的数据相比对，均未发现此7种突变，而在706例正常男性中也未发现这7种新的变异。
1.2.3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4对引物对仅在非梗阻性无精子症患者(W024，W033，W251，W505，W520，W566，W570，W628，W698)DAX-1基因存在的6个错义突变位点进行特异性扩增，DAX-1序列从NCBI数据库中查得。引物由Invitrogen公司合成，所合成的4种引物见表1。DAX-1基因在NCBI数据库中的编号是GeneID:190。采用primer1引物对的PCR产物的序列如SEQIDNO:23所示；采用primer2引物对的PCR产物的序列如SEQIDNO:24所示；采用primer3引物对的PCR产物的序列如SEQIDNO:25所示；采用primer4引物对的PCR产物的序列如SEQIDNO:26所示。
表1DAX-1点突变验证引物
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3μlPCR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。
1.2.4质粒构建
1.2.4.1DAX-1野生型表达质粒的构建
1.2.4.1.1人DAX-1基因cDNA编码区全长序列的获取
将人睾丸组织RNA逆转录合成cDNA，并以此为模板，设计含有HindⅢ和BamH1两种限制性酶切位点的引物，以3’带HA多肽标签的pcDNA3.1(+)为载体，构建野生型DAX-1表达质粒。带有酶切位点的引物序列为：
上游：5'-CCCAAGCTTATGGCGGGCGAGAAC-3'(SEQIDNO:9)
下游：5'-CGCGGATCCCGTATCTTTGTACAG-3'(SEQIDNO:10)
其中上游引物5’端含有HindⅢ酶切位点，下游引物5’端含有BamH1酶切位点。1.2.4.2DAX-1突变体表达质粒的构建：
以测序正确的pcDNA3.1-DAX1为模板，使用设计合成的6对点突变引物，构建DAX-16种突变体表达质粒。引物由Invitrogen公司合成，所合成的6对引物见表2。
表2DAX-16对定点突变引物
1.2.5双荧光素酶报告基因实验
1.2.5.2质粒准备
野生型DAX-1表达载体：pcDNA3.1-DAX-1WT(WT组)
突变型DAX-1表达载体：pcDNA3.1-DAX-1R51K(R51K组)
pcDNA3.1-DAX-1C104W(C104W组)
pcDNA3.1-DAX-1A242V(A242V组)
pcDNA3.1-DAX-1E256Q(E256Q组)
pcDNA3.1-DAX-1V385L(V385L组)
pcDNA3.1-DAX-1I427V(I427V组)
1.2.5.3HeLa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。
1.2.5.4HeLa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LipofectamineTM2000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Lipofectamine2000对细胞的毒性。
1.2.5.5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PBS1ml洗涤2次；
2)将5×PassiveLysisBuffer稀释成1×PassiveLysisBuffer，用移液枪向每孔加入100μlPassiveLysisBuffer；
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19μlLuciferaseAssayⅡBuffer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19μlSTOPBuffer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。
1.2.6免疫共沉淀实验
1.2.6.2质粒准备
人AR表达质粒：pcDNA3.1-AR
野生型DAX-1表达载体：pcDNA3.1-DAX-1WT(WT组)
突变型DAX-1表达载体：pcDNA3.1-DAX-1R51K(R51K组)
pcDNA3.1-DAX-1C104W(C104W组)
pcDNA3.1-DAX-1A242V(A242V组)
pcDNA3.1-DAX-1E256Q(E256Q组)
pcDNA3.1-DAX-1V385L(V385L组)
pcDNA3.1-DAX-1I427V(I427V组)
1.2.6.3HeLa细胞培养
将HeLa细胞用含10％胎牛血清高糖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。
1.2.6.4细胞总蛋白的提取
用含有10％胎牛血清的DMEM完全培养基培养HeLa细胞，放置于37℃、5％CO2的培养箱中，按照无菌操作台内的常规细胞培养方法培养。将处于对数生长期的HeLa细胞以3×105/孔密度接种于6孔培养板，待第二天细胞贴壁后，根据转染试剂LipofectamineTM2000的转染步骤，将重组质粒pcDNA3.1(+)-3，HA-DAX1WT及6种突变体mut1-mut6(实验组，2ug)和pcDNA3.1(+)-3，HA空载(对照组，2ug)与hAR的表达载体(2ug)分别共转染到HeLa细胞。转染48h后吸弃培养基，根据Qiagene公司的哺乳动物蛋白提取试剂盒的提取步骤用1×PBS溶液洗涤2次，加入1ml预冷的PBS用细胞刮轻轻挂下，转移到1.5ml的Ep管，450×g，4℃离心5min，弃掉上清置于冰上，用100ulLysisBuffer(含0.1UBenzonaseNuclease,1ul100×ProteaseInhibitor)重悬沉淀，4℃旋转5min后，14,000×g,4℃离心10min，将上清转移至新的离心管中。
1.2.6.5免疫共沉淀
将实验组与对照组提取的蛋白平均分成4分，其中一份作为Input样品，加入6.25ul的5×SDSLoadingBuffer(含5％1mMDTT)，混匀后98℃处理10min，-80℃保存。余下3份上清液均加入475ul的LysisBuffer或Co-IPBuffer，补充至每管总体积为500ul，之后分别加入2ugAR抗体，2ugHA抗体，2ugIgG；4℃旋转1h；每管加入50ul预处理后的ProteinA/GBeads4℃旋转过夜。
4℃旋转过夜后12,000×g，4℃瞬时离心20s，弃上清；加入600ulLysisBuffer重悬,12,000×g，4℃离心1min洗涤Beads(不用枪头吸，用手弹起即可)，共洗涤3次，用枪吸出上清,但不要吸干，注意不能吸到Beads；向Beads中加入15ul的LysisBuffer和等体积2XSDS上样缓冲液，混匀后98℃处理10min，将蛋白即抗原抗体复合物从Beads上解离下来，即可用于蛋白电泳。制备SDS聚丙烯酰胺凝胶，每孔上样量15ul，常规电泳后将蛋白转移至硝酸纤维素膜上，用含有5％脱脂奶粉的TBS-T缓冲液(0.02MTris-Cl，pH7.6，0.137MNaCl，0.1％Tween-20)室温封闭1小时后，弃去封闭液。加入一定量适当稀释于含5％脱脂奶粉的TBS-T缓冲液中的识别目的蛋白的一抗(鼠抗人HA和兔抗人AR单克隆抗体，1:2000稀释)，4℃过夜；弃去一抗，TBS-T缓冲液清洗3次，每次5分钟。加入一定量适当稀释于含5％脱脂奶粉的TBS-T缓冲液中的二抗(1:10000稀释)，室温孵育1小时；弃去二抗，TBS-T缓冲液清洗3次，每次5分钟。在膜上滴加ECL化学发光底物，然后用X光片曝光，经冲片机自动显影定影。
1.2.7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非梗阻性无精子症患者DAX-1基因点突变的鉴定
对776例非梗阻性无精子症患者和706例正常可育男性的DAX-1基因的外显子测序结果分析，如表3所示：DAX-1基因的7个新突变均为纯合突变，突变位点分别为：c.152G>A(p.R51K)、c.312C>G(p.C104W)、c.725C>T(p.A242V)、c.766G>C(p.E256Q)、c.1153G>T(p.V385L)、c.1279A>G(p.I427V)和c.162G>A(p.L54L)，前6个位错义突变，最后1个为同义突变，与dbSNP135数据库和千人基因组数据库中的数据相比对，均未发现有这些突变，而在706例正常可育男性中也未发现这些新的变异(如表3所示)。进一步的PCR验证错义突变的结果如图1所示。
表3DAX-1点突变在非梗阻性无精子症组和正常组中的情况
注：病人编号以W开头，仅编号7为同义突变。
2.2DAX-1野生型及突变体与AR在细胞内的结合作用
为了进一步检测DAX-1突变后与AR的相互作用情况，采用免疫共沉淀技术，在HeLa细胞中分别共转入hAR和DAX-1野生型及突变体表达质粒，转染48h后收集细胞提取总蛋白，做anti-AR和anti-HA的免疫共沉淀以及anti-AR和anti-HA的WesternBlot检测蛋白的相互作用和表达情况，结果显示与DAX-1野生型相比，仅DAX-1V385L突变体与AR的结合减弱。
2.3DAX-1突变体与AR相互作用后对AR下游靶基因MMTV表达的影响
DAX-1作为辅因子与AR相互作用来调控下游基因的表达，为了进一步检测DAX-16种突变体与AR相互作用后对AR下游靶基因MMTV表达的影响，采用双荧光素酶报告基因实验，将AR表达质粒和MMTV-LUC与DAX-1野生型及6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DAX-1一致，DAX-1R51K,C104W,A242V,E256Q,I427V突变体也可以明显抑制MMTV启动子的活性，仅DAX-1V385L突变体与野生型有显著差异，不能抑制MMTV启动子的活性，如图3所示。
3讨论
DAX-1基因突变主要表现为肾上腺皮质发育不全，伴或不伴有低促性腺激素性性腺功能减退。由于多数患者首发症状典型，确诊并不容易。DAX-1基因为孤儿核受体，在肾上腺皮质、性腺、下丘脑和垂体中表达。DAX-1基因含2个外显子，共编码470个氨基酸。1994年Muscatelli等首次报道DAX-1单基因突变与AHC和HH突变有关，上海瑞金医院2007年在国内首次报道了此病患者。DAX-1基因突变类型的发生频率依次为单独或伴有邻近基因共同缺失(约40％)、移码突变(约28％)、无义突变(约18％)和错义突变(约14％)。多数突变位点位于推测的配体结合区域，致使其转录功能受损。本实验研究发现7个DAX-1基因点突变中有6个为错义突变，1个为同义突变，且与dbSNP135数据库和千人基因组数据库中的数据相比对，均未发现有这些突变。Holter等人的研究表明，雄激素受体AR是DAX-1的一个新的作用靶点，DAX-1通过与AR直接相互作用抑制AR的转录活性。本实验通过构建DAX-1野生型及突变体表达质粒，研究DAX-1在细胞中的功能。通过测序结果及功能研究分析DAX-1野生型及突变体表达质粒构建成功，DAX-1V385L突变体的功能与DAX-1野生型相比有显著性差异。
DAX-l在下丘脑、垂体、肾上腺和性腺等组织中均有表达。免疫组化证实：从第11.5天开始，DAX-l在胚鼠前脑中表达，第13.5天在垂体前叶中表达，第l4.5天在间脑腹侧表达，第l8天在丘脑下部的腹内侧核中表达。且研究证实小鼠性腺发育的不同时期均有DAX-l的表达：从第9天胚鼠出现尿生殖嵴时开始表达DAX-1；性别分化后，在雄性小鼠睾丸中DAX-l的水平先迅速下降，然后逐渐升高，第l2天在Sertoli细胞和Leydig细胞中达到高峰，推测DAX-l在睾丸发育晚期可能具有调控雄性特异性基因的作用；但在雌性小鼠中则不同，第l3.5天时性腺中仍有表达，并持续到成年，但作用不详。人DAX-1基因位于Xp2l，人与鼠DAX-1基因同源性为65％。它是不典型的孤核受体家族成员，缺乏DNA结合域，在脊椎动物孤核受体家族中属于第0类。DAX-l也有一个保守的LBD，它的N端重叠区是一个锌指的保守序列，可与DNA上的发夹结构结合。核受体通常均有保守的LBD，通过与配体结合可激活核受体的靶基因。DAX-l可能是进化过程中出现较早的核受体家族，在演变过程中LBD发生突变，失去与配体结合的能力，因此虽然DAX-l有LBD，但目前尚未找到它的配体。
本实验通过对临床资料的收集，利用大规模测序技术在非梗阻性无精子症病人中筛选DAX-1基因的突变位点，功能试验结果显示DAX-1V385L突变体及DAX-1野生型在细胞内的功能有显著差异，提示这个突变位点与非梗阻性无精症的发生存在相关性，DAX-1可能是临床男性不育一个潜在的诊疗靶点。
4结论
4.1通过大规模测序筛选出仅在非梗阻性无精子症患者中DAX-1基因的6个错义突变，其相应的氨基酸改变为：R51K、C104W、A242V、E256Q、V385L、I427V，与dbSNP135数据库和千人基因组数据库中的数据相比对，均未发现有这些突变。
4.2DAX-1野生型及DAX-1V385L突变体与AR在细胞内的结合作用有显著差异。
4.3DAX-1V385L突变体与AR相互作用后对AR下游靶基因MMTV的表达与DAX-1野生型相比有显著性差异。
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。
Claims (3)
1.一种检测与非梗阻性无精子症相关的遗传标记的引物对，其特征在于，所述遗传标记的核苷酸序列是DAX-1基因的序列，所述序列的突变位点选自c.152G>A、c.312C>G、c.725C>T、c.766G>C、c.1153G>T、c.1279A>G和c.162G>A中的一个或多个；所述引物对，选自primer1、primer2、primer3或primer4引物对，其中，
所述primer1引物对的上游引物的序列如SEQIDNO:1所示，其下游引物的序列如SEQIDNO:2所示；
所述primer2引物对的上游引物的序列如SEQIDNO:3所示，其下游引物的序列如SEQIDNO:4所示；
所述primer3引物对的上游引物的序列如SEQIDNO:5所示，其下游引物的序列如SEQIDNO:6所示；
所述primer4引物对的上游引物的序列如SEQIDNO:7所示，其下游引物的序列如SEQIDNO:8所示。
2.根据权利要求1所述的检测与非梗阻性无精子症相关的遗传标记的引物对，其特征在于，所述遗传标记的核苷酸序列是DAX-1基因的序列，所述序列的突变位点选自c.152G>A、c.312C>G、c.725C>T、c.766G>C、c.1153G>T和c.1279A>G中的一个或多个。
3.根据权利要求2所述的检测与非梗阻性无精子症相关的遗传标记的引物对，其特征在于，所述突变位点是c.1153G>T。
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| CN105506110A (zh) * | 2015-12-31 | 2016-04-20 | 杭州艾迪康医学检验中心有限公司 | 检测tex11基因第8、25号全外显子的方法和引物 |
| CN107227370A (zh) * | 2017-07-24 | 2017-10-03 | 胡松 | 染色体基因引物对和探针组合以及染色体核酸检测试剂盒 |
| CN109943631A (zh) * | 2019-03-21 | 2019-06-28 | 吉林省银丰生物工程技术有限公司 | 非梗阻性无精子症辅助诊断基因检测试剂盒 |
| CN113278689B (zh) * | 2021-04-20 | 2022-05-03 | 厦门市妇幼保健院（厦门市计划生育服务中心） | Atg4d作为靶标在诊断和治疗非梗阻性无精子症中的应用 |
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| DONATA PONIKWICKA-TYSZKO等: "The DAX1 mutation in a patient with hypogonadotropic hypogonadism and adrenal hypoplasia congenita causes functional disruption of induction of spermatogenesis", 《J ASSIST REPROD GENET》 * |
| ZANARIA E.等: "登录号：S74720", 《GENBANK》 * |
| 叶勤: "《现代生物技术原理及其应用》", 1 August 2003 * |
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| CN105176976B (zh) | 2018-09-25 |
| CN104531683A (zh) | 2015-04-22 |
| CN104531683B (zh) | 2017-11-07 |
| CN105112501B (zh) | 2018-04-10 |
| CN105112501A (zh) | 2015-12-02 |
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