CN107164520A

CN107164520A - 一种严重弱精子症新致病基因及其应用

Info

Publication number: CN107164520A
Application number: CN201710495499.1A
Authority: CN
Inventors: 沙艳伟; 李萍; 李琳; 张玲; 许晓慧; 邓冰冰; 何雪梅; 林津; 叶雅萍; 高海杰; 陈静; 王雄
Original assignee: BEIJING OBSTETRICS AND GYNECOLOGY HOSPITAL CAPITAL MEDICAL UNIVERSITY; Xiamen Maternity & Child Healthcare Hospital (xiamen City Family Planning Service Center); Yantai Yuhuangding Hospital
Current assignee: BEIJING OBSTETRICS AND GYNECOLOGY HOSPITAL CAPITAL MEDICAL UNIVERSITY; Xiamen Maternity & Child Healthcare Hospital (xiamen City Family Planning Service Center); Yantai Yuhuangding Hospital
Priority date: 2017-06-26
Filing date: 2017-06-26
Publication date: 2017-09-15

Abstract

一种严重弱精子症新致病基因及其应用，涉及致病基因，首次发现一种严重弱精子症新致病基因SPAG17。以严重弱精子症一个患病家系及患病个体为研究对象，对家系中的患病个体进行了外显子组测序和比较，发现患者SPAG17基因存在基因突变。利用这个突变可以对严重弱精子症进行检测。

Description

一种严重弱精子症新致病基因及其应用

技术领域

本发明涉及致病基因，尤其是涉及一种严重弱精子症新致病基因及其应用。

背景技术

严重弱精子症是一种较常见的导致男性不育的畸形精子症，是一种与遗传、生活方式等因素有关的疾病，其主要临床特征是精子运动能力的严重丧失。国外早期的分子方面发病机制研究发现AKAP3、AKAP4、SLC26A8、DNAH1、CATSPER2、GALNTL5等基因发生了突变(1.Takasaki N,Tachibana K,Ogasawara S,Matsuzaki H,Hagiuda J,Ishikawa H,Mochida K,Inoue K,Ogonuki N,Ogura A,Noce T,Ito C,Toshimori K and NarimatsuH.A heterozygous mutation of GALNTL5affects male infertility with impairmentof sperm motility.Proc Natl Acad Sci U S A.2014；111(3):1120-1125；2.Dirami T,Rode B,Jollivet M,Da Silva N,Escalier D,Gaitch N,Norez C,Tuffery P,Wolf JP,Becq F,Ray PF,Dμlioust E,Gacon G,Bienvenu T and Toure A.Missense mutations inSLC26A8,encoding a sperm-specific activator of CFTR,are associated with humanasthenozoospermia.Am J Hum Genet.2013；92(5):760-766.)，并导致精子尾部形态及运动异常。

目前严重弱精子症的基因突变谱尚未完全发现，基因型和表现型的关系也不明确。目前单基因疾病的研究开始大量采用全外显子组测序(whole-exome sequencing)及全基因组测序(whole-genome sequencing)的方法，这两种方法被成功的应用于发现稀有单基因疾病的致病基因。全外显子组测序及全基因组测序技术已被证明为降低稀有单基因疾病候选基因甚至发现其致病基因的有力、有效手段。仅通过对几个很少的个体(包括患者及正常对照)的全外显子组或基因组进行测序来筛选与疾病相关的变异，其成功率大为提升。

因此本领域对严重弱精子症的研究尚不清晰，对造成该疾病的原因更不明了，因此本领域迫切需要对严重弱精子症的致病机理进行研究，找到新致病基因及突变位点。

发明内容

本发明的第一目的在于提供通过外显子组测序的方法确定严重弱精子症的新致病基因。

本发明的第二目的在于提供严重弱精子症的生物标记物。

本发明的第三目的在于提供突变的SPAG17基因。

本发明的第四目的在于提供突变的SPAG17蛋白。

本发明的第五目的在于提供一种检测严重弱精子症的方法。

本发明的第六目的在于提供通过PCR检测SPAG17基因或SPAG17蛋白突变中使用的引物对。

本发明的第七目的在于提供与突变SPAG17基因互补的核酸探针。

本发明的第八目的在于提供检测突变SPAG17基因或SPAG17蛋白的试剂盒。

本发明的第九目的在于提供检测突变SPAG17基因的试剂盒。

本发明的第十目的在于提供一种严重弱精子症新致病基因的应用。

所述通过外显子组测序的方法确定严重弱精子症的新致病基因。

所述严重弱精子症的生物标记物，即突变的SPAG17基因或SPAG17蛋白，所述生物标记物是具有选自如下的突变的SPAG17基因或SPAG17蛋白：

在外显子30中错义突变(c.4343G>A；p.R1448Q)。

所述突变的SPAG17基因为SEQ ID NO:1的序列中具有以下突变：

在外显子30中错义突变(c.4343G>A)。

所述突变的SPAG17蛋白为SEQ ID NO:2的序列中具有以下突变：

在外显子30中错义突变(p.R1448Q)。

所述一种检测严重弱精子症的方法，包括检测受试者的SPAG17基因或SPAG17蛋白中是否存在突变位点，若有突变位点，则所述受试者被鉴定为患有严重弱精子症或易患严重弱精子症，所述突变位点选自如下一种：

在外显子30中错义突变(c.4343G>A；p.R1448Q)。

所述SPAG17蛋白为SEQ ID NO:2的序列表示。

所述SPAG17基因为SEQ ID NO:1的序列表示。

所述检测严重弱精子症的方法包括如下至少一组引物扩增的步骤：

SEQ ID NO:3和SEQ ID NO:4。

所述检测严重弱精子症的方法中检测突变位点通过选自如下进行：测序、电泳、核酸杂交、原位杂交、PCR、逆转录酶链反应和变性高效液相色谱。

所述通过PCR检测SPAG17基因或SPAG17蛋白突变中使用的引物对，所述突变是选自如下一种：

在外显子30中错义突变(c.4343G>A；p.R1448Q)，

其中所述引物对分别基于选自如下的位置前后设计，使得扩增该位置(编号基于SPAG17的cDNA序列)：4343。

所述与突变SPAG17基因互补的核酸探针，所述突变是选自如下一种：

在外显子30中错义突变(c.4343G>A；p.R1448Q)，

所述探针与突变SPAG17基因的互补区包括选自如下的位置(编号基于SPAG17的cDNA序列)：4343。

所述检测突变SPAG17基因或SPAG17蛋白的试剂盒，包含一组或多组引物对，其中所述突变是选自如下一种：

在外显子30中错义突变(c.4343G>A；p.R1448Q)，

其中所述引物对分别基于选自如下的位置前后设计，使得其扩增产物涵盖该位置(编号基于SPAG17的cDNA序列)：4343。

所述检测突变SPAG17基因或SPAG17蛋白的试剂盒包含选自如下的至少一组引物：

SEQ ID NO:3和SEQ ID NO:4。

所述检测突变SPAG17基因的试剂盒，包含一个或多个核酸探针，所述突变是选自如下一种：

在外显子30中错义突变(c.4343G>A；p.R1448Q)，

所述探针与突变SPAG17基因上包含选自如下的位置的区域互补(编号基于SPAG17的cDNA序列)：4343。

所述一种严重弱精子症新致病基因的应用。

本发明将为严重弱精子症的发病机制研究奠定重要基础，有可能为严重弱精子症的患者治疗提供全新的理论依据。

具体实施方式

在本发明中，采用本领域通用表示法表示突变。例如，突变(c.4343G>A；p.R1448Q)中，c表示cDNA，p表示蛋白质，DNA水平的突变对应蛋白质水平的突变。

野生型SPAG17基因的cDNA序列如SEQ ID NO:1所示。

SEQ ID NO:2：野生型SPAG17蛋白的氨基酸序列。

提及基因序列，本领域技术人员应当理解，实际包括互补双链的任意一条，或者两条。虽然多数情况下至给出了一条链，但实际上也公开了与之互补的另一条链。例如提及SPAG17基因的cDNA序列，实际上包括该序列以及其互补序列。例如，提及SEQ ID NO:1，实际包括其互补序列。本领域技术人员还可以理解，利用一条链可以检测另一条链，反之亦然。

本申请中的基因序列包括DNA形式或RNA形式，公开其中一种，意味着另一种也被公开。例如提及SPAG17基因的cDNA序列，实际也包括相应的RNA序列。

实施例1确定严重弱精子症的致病基因。

本发明收集1个近亲婚配家系中的两位双胞胎患有严重弱精子症的病例，两位患者均表现为不育。先证者29岁，父母为三代内姑表亲结婚，生育3男1女，哥哥生育1子，妹妹生育两胎，双胞胎弟弟不育。先证者外生殖器发育正常，双侧睾丸大小正常，双侧精索静脉未触及异常。外周血染色体核型分析正常，Y染色体微缺失检测未发现异常。无支气管扩张等上呼吸道疾病，无内脏转位，无鼻窦炎病史。在我院三次精液检查平均值为精液量2.8ml，精子密度27.5百万/ml，前向运动精子占比1.8％，非前向运动精子1.6％。精子形态改良巴氏染色读取显示正常形态精子4.5％，精子顶体酶28mIU/ml，精浆生化果糖、中性糖苷酶活性及精浆锌均未见异常，根据以上检查结果提示患者为严重弱精子症。

本发明对先症者进行了全外显子组测序，具体步骤如下：

样品制备：采集所述患者及其父母家人外周血，利用试剂盒抽提外周血白细胞中的基因组DNA(QIAamp DNA Mini Kit 51304.Qiagen,USA)，利用NanoDrop 2000测量DNA的浓度及纯度(Thermo Scientific,USA)，所得的每个标本基因组DNA的OD260/OD280均位于1.7～2.0之间，浓度不少于100ng/μl，总量不少于30μl。

然后，对上述1个样本的外显子组序列进行了测序。测序平台为Illumina Hiseq2000，依照Illumina标准建库说明书(参见http://www.illumina.com/)进行测序，简述如下：

1)使用Illumina公司试剂盒TruSeq DNA Library Prep Kit制作DNA文库。在外显子捕获过程中使用Agilent公司试剂盒SureSelect Human All Exon V5；

2)使用Illumina公司HiSeq2000测序平台对捕获的外显子区域进行平行测序读取长度为90bp，每个样本的平均测序深度最少为100；

3)使用Burrows-Wheeler Aligner(http://bio-bwa.sourceforge.net/)将测序结果比对到人参考基因组中(hg19)。使用SAMtools将重复读取结果去除，通过GATK(https://www.broadinstitute.org/gatk/)进行调整和校准。然后使用SAMtools(http://www.samtools.sourceforge.net/)挖掘出SNVs及InDels，并用ANNOVAR(http://www.openbioinformatics.org/annovar/)命名。

将等位基因频率大于1％的突变位点剔除(参考数据库包括dbSNP，1000Genomes，Exome Aggregation Consortium(ExAC))。

发现先症者在基因SPAG17上具有纯合突变：通过家系中2名患者和4位正常人SNP筛查共分离，这些位点可能是致病位点。

结果显示于表3。

因此本发明认为SPAG17基因极有可能为严重弱精子症的致病基因。

实施例2在其他病例中确认上述严重弱精子症的致病基因。

本发明纳入所有严重弱精子症患者，对SPAG17基因为严重弱精子症的致病基因进行验证。验证中利用Sanger法测序验证SPAG17基因的突变，其中考虑了家系内等样本验证情况。

分别对2名患者、4名家系内正常人(即其中2名患者的父母及哥哥姐姐，他们均未发病)基因进行检测。

具体方法步骤如下：

1.DNA提取：

分别提取2名患者、4名家系内正常人的外周静脉血，按照实施例1的方法提取基因组DNA和测定DNA含量。

2.引物设计及PCR反应

引物设计参考人类基因组序列数据库hg19，引物序列参见表1。

表1

PCR扩增体系参见表2。

表2

PCR反应条件：

94℃变性5min，然后进入每个循环，即94℃变性30s，退火(通常为55℃，根据不同引物的退火温度设定)30s，72℃延伸1min，总共设定30个循环。所有循环结束，最终72℃延伸10min，并将PCR产物保存在4或-20℃。

3)将步骤2)中获得的获自2名患者、4名家系内正常人(即该2例患者的父母，他们均未发病)的PCR扩征产物直接进行DNA测序，方法同实施例1。

本发明收集到的家系内2例严重弱精子症病例均在SPAG17基因中检测到有意义的突变位点。同时所发现的突变位点在家族正常人中均未能检测到。这些突变位点在ExAC数据库中的频率极低或无频率(见表3)，提示所检测到的位点很可能并非SNP。因此认为SPAG17基因为严重弱精子症的致病基因。

表3

序列表说明

SEQ ID NO:1：野生型SPAG17基因的cDNA序列

ATGGCACCCAAGAAGGAGAAAGGAGGAACTGTGAACACCAGTTCTAAGATATGGGAACCCTCGCTCATAGCTGCACAGTTCAATCAGAACGATTGGCAGGCCTCCATTGCTTTTGTGGTTGGGAACCAGATTGAAGATGATCTTCTCATCCAAGCCCTTACCGTGGCTGTCCAGGTCCCTCAGCGTAAACTCTTCAGTATGGTGTCGTGGCAAGACATTCTCCAGCAGATTAATGAAATAAATACACTTGTTGGATCTGCTTCATCTAAAAAGGCAAAAAAACCTGTAGGTGGTAATGCTCCTTTATATTATGAGGTGTTAACGGCAGCAAAAGCAATTATGGATAGTGGAGAGAAATTAACCTTACCACTGATAGGGAAACTCTTGAAATTTCAACTTCTCCAGATTAAATTTAAGGACCAACAGCGACGGGAAAATGAAAAGAAGGTAATAGAAGACAAACCTAAGTTAGAAAAGGATAAAGGGAAAGCAAAATCTCCCAAGGAGAAAAAGGCTCCAAGTGCCAAGCCTGCCAAAGGAAAGGGAAAGGATCAGCCTGAGGCAAATGCACCAGTGAAAAAGACCACCCAGTTAAAGCGGAGAGGAGAAGACGACCACACCAATCGTTACATTGACGATGAGCCAGATGATGGTGCCCAACATTACATTATAGTTGTGGGCTTTAACAATCCTCAGCTATTAGCAATTATGGCTGAGCTTGGCATTCCTATAACCAGCGTGATTAAAATATCTTCAGAGAATTATGAACCTCTGCAGACACACCTGGCAGCAGTTAACCAGCAGCAGGAAGTTCTTCTTCAGTCAGAAGATCTAGAAGCAGAAAAATTGAAGAAAGAAAATGCCATAAAAGAGCTTAAAACTTTCTGGAAGTACTTGGAACCAGTCCTGAATAATGAGAAACCTGAAACAAATCTCTTTGATGTTGCTCGACTTGAGTACATGGTCAAAGCAGCTGATTTTCCTTCTGACTGGTCAGATGGTGAGATGATGCTGAAATTGGGCACTGATATTTTTGAAAATATTGCCTGCTTGATGTATGACATCCTGGATTGGAAAAGGCAGCACCAGCACTATTTGGAAAGCATGCAGCTTATTAATGTTCCACAAGTGGTTAATGAGAAACCTGTATTAGAAGCCATGCCAACTTCAGAGGCTCCACAACCTGCTGTACCAGCTCCTGGAAAGAAGAAAGCACAGTATGAAGAACCGCAAGCTCCACCACCAGTGACTTCAGTCATCACAACTGAAGTAGACATGAGATATTACAATTATTTGCTGAATCCAATTCGAGAGGAATTCATTTCTGTGCCCCTGATACTGCATTGTATGCTGGAACAGGTTGTTGCAACTGAAGAAGATCTCGTCCCACCCAGTCTGCGGGAGCCATCCCCCAGAGCAGACGGGCTAGACCACAGAATCGCAGCTCACATTGTGTCCCTTCTGCCCTCACTCTGTCTCTCAGAGAGGGAGAAAAAGAATCTTCATGACATATTTTTATCTGAAGAAGAAAATGAAAGCAAAGCAGTGCCCAAAGGCCCCCTCCTACTGAACTATCATGATGCACACGCCCACAAGAAGTACGCACTACAGGACCAAAAGAATTTTGATCCAGTTCAAATTGAGCAGGAGATGCAGTCCAAGTTGCCACTGTGGGAATTTCTTCAATTCCCTCTACCCCCACCATGGAACAACACTAAACGTCTAGCTACAATTCATGAGCTTATGCACTTTTGTACGAGTGATGTCTTGAGCTGGAATGAAGTAGAACGAGCCTTCAAGGTGTTTACTTTTGAGAGCCTGAAGCTCTCTGAGGTTGATGAAAAAGGGAAACTGAAACCTTCTGGGATGATGTGTGGGTCAGATTCTGAAATGTTCAACATACCGTGGGACAACCCTGCCAGATTTGCTAAACAGATAAGGCAGCAATATGTCATGAAAATGAATACTCAAGAGGCCAAGCAGAAAGCAGATATTAAAATCAAAGACAGAACACTATTTGTGGATCAGAATTTGTCAATGTCTGTGCAAGATAATGAAAGCAACCGAGAACCTTCAGATCCTAGTCAGTGTGATGCTAACAATATGAAGCATTCTGACTTGAATAATCTCAAACTCTCAGTCCCTGATAATAGACAGCTGTTAGAGCAGGAGAGCATCATGAAGGCTCAGCCCCAACATGAGTCTCTGGAGCAGACCACAAACAATGAGATCAAAGATGATGCAGTCACAAAGGCTGATTCTCATGAAAAGAAACCCAAGAAGATGATGGTGGAAGCAGATTTAGAGGACATAAAGAAAACACAGCAGCGCAGTCTAATGGACTGGAGTTTTACTGAACATTTTAAACCGAAAGTACTGCTTCAGGTCCTTCAAGAAGCCCATAAGCAATATAGGTGTGTTGATTCTTACTACCACACCCAAGACAACTCTTTACTTTTAGTCTTTCACAATCCAATGAATAGACAACGTTTGCATTGTGAATATTGGAACATTGCTCTCCACTCCAATGTTGGATTCAGGAATTATTTGGAACTTGTTGCAAAATCTATTCAAGATTGGATTACAAAAGAAGAAGCTATATATCAGGAATCTAAAATGAATGAGAAAATCATCAGGACCAGAGCTGAGCTGGAATTGAAATCTTCTGCTAATGCCAAACTTACTTCTGCTAGCAAAATTTTTTCCATTAAAGAATCTAAAAGTAACAAAGGAATCAGCAAAACAGAGATATCAGATCAAGAAAAAGAAAAAGAGAAGGAAAAGATTCCTTTCATTTTAGAAGGCTCTCTCAAGGCATGGAAAGAAGAGCAACATCGATTAGCAGAAGAGGAGCGCTTAAGGGAAGAAAAGAAAGCAGAGAAGAAGGGTAAAGAAGCTGGTAAAAAGAAAGGCAAGGATAACGCAGAGAAAGAGGATAGTAGGTCTTTGAAGAAAAAATCACCTTACAAGGAGAAATCTAAAGAAGAACAAGTCAAGATCCAAGAAGTAACAGAAGAGTCCCCCCACCAACCAGAACCTAAGATAACTTACCCGTTTCACGGATACAATATGGGAAATATACCCACTCAAATCTCAGGGTCAAATTACTACCTGTATCCTTCTGATGGGGGGCAGATTGAAGTGGAAAAGACAATGTTTGAAAAAGGCCCAACTTTTATCAAAGTGAGAGTGGTAAAGGACAACCACAATTTTATGATTCATTTAAATGACCCTAAGGAAATTGTGAAAAAGGAAGAGAAAGGGGATTATTATTTAGAAGAGGAAGAAGAAGGAGATGAGGAACAAAGTCTTGAAACGGAAGTATCAGATGCAAAGAATAAAGCTTTCAGCAAGTTTGGATCTTTTTCTGCCACCTTAGAAAATGGAATCTGCCTCTCGATAAGTTACTATGGATCAAATGGAATGGCACCAGAAGATAAGGATCCTGATTTAGAAACAATATTGAATATCCCTTCAGCACTCACTCCAACAGTGGTTCC

TGTTATAGTGACCGTTCCTCAAAGCAAAGCTAAAGGGAAAATAAAAGGCAAAGAAAAACCCAAAGAATCCCTTAAAGAAGAAGAACACCCAAAAGAAGAAGAGAAAAAGGAAGAAGAAGTAGAACCAGAACCTGTTTTACAAGAGACTTTGGATGTTCCCACCTTCCAGAGCCTAAATGTGTCTTGCCCCAGTGGGCTCCTGTTGACTTTCATTGGACAAGAATCTACAGGTCAATATGTTATAGATGAGGAACCCACCTGGGACATCATGGTCCGTCAGAGCTACCCCCAGAGGGTGAAGCACTATGAGTTCTATAAAACGGTGATGCCACCCGCAGAGCAGGAGGCTTCAAGGGTTATCACCAGTCAAGGCACTGTTGTCAAATATATGTTGGATGGATCCACACAGATTCTCTTTGCAGATGGTGCTGTGAGCAGGAGTCCCAATTCAGGTCTTATTTGTCCTCCTTCTGAAATGCCAGCAACGCCTCACAGTGGAGATTTGATGGACTCTATTTCTCAGCAGAAATCAGAAACGATACCATCTGAGATTACCAACACAAAGAAAGGAAAAAGTCACAAAAGTCAGTCATCAATGGCCCATAAGGGTGAAATCCATGACCCTCCTCCAGAGGCAGTTCAAACTGTAACTCCTGTGGAGGTTCACATAGGCACCTGGTTTACAACCACACCTGAAGGAAATCGGATCGGCACCAAAGGATTAGAAAGAATAGCAGACTTGACCCCATTGTTATCCTTTCAGGCCACAGATCCTGTCAATGGAACGGTTATGACAACTCGAGAAGACAAAGTTGTCATAGTTGAAAGGAAAGATGGTACTCGGATAGTGGATCATGCTGATGGTACCAGAATCACAACCTTTTATCAAGTTTATGAAGATCAAATTATTCTGCCAGATGATCAAGAAACAACCGAGGGTCCTCGGACTGTCACCAGGCAGGTGAAGTGTATGCGGGTAGAAAGCTCACGCTATGCCACTGTTATCGCCAACTGTGAGGACAGTAGCTGCTGTGCCACCTTTGGAGATGGAACAACTATTATTGCAAAGCCACAGGGAACATACCAGGTGTTACCTCCAAACACAGGCTCTCTTTATATTGACAAGGATTGTTCAGCTGTGTACTGCCATGAGTCAAGCAGTAATATATACTATCCTTTTCAAAAGCGTGAGCAGCTGCGAGCTGGCAGGTACATCATGAGGCATACTTCAGAGGTTATCTGTGAGGTTCTGGATCCTGAGGGAAACACTTTTCAGGTCATGGCTGATGGTAGCATATCAACTATATTACCTGAAAAAAAATTGGAAGATGATTTAAATGAGAAAACTGAGGGCTATGATAGTCTGTCCTCTATGCACCTTGAAAAGAATCATCAGCAAATCTATGGTGAACATGTCCCCAGGTTTTTTGTTATGTATGCTGATGGATCAGGAATGGAACTTCTTCGAGACAGTGACATAGAAGAATATCTATCTTTGGCATATAAAGAATCAAATACTGTTGTTCTCCAAGAGCCAGTGCAGGAACAGCCAGGCACCCTAACCATCACAGTCCTTCGCCCTTTCCATGAAGCATCACCATGGCAAGTAAAAAAGGAAGATACAATTGTCCCTCCTAATCTCCGGTCAAGGTCATGGGAAACATTTCCCTCAGTTGAGAAAAAAACTCCAGGACCTCCGTTTGGTACTCAGATTTGGAAAGGCCTTTGCATTGAGTCCAAACAGCTAGTGAGTGCCCCGGGTGCCATACTCAAGAGCCCCAGTGTGCTACAGATGCGCCAATTCATTCAGCATGAGGTCATAAAGAATGAGGTGAAACTGAGGCTGCAGGTTTCCCTTAAGGATTACATAAACTATATTCTAAAGAAAGAAGATGAGCTGCAGGAAATGATGGTTAAAGATTCCAGAACTGAGGAGGAGAGAGGCAATGCTGCTGATCTCCTCAAGCTGGTTATGTCTTTCCCTAAAATGGAGGAAACTACAAAAAGTCATGTTACTGAAGTTGCAGCTCACCTAACTGATTTATTCAAGCAGTCTTTGGCTACGCCTCCAAAATGCCCACCAGACACATTTGGTAAAGATTTCTTTGAAAAGACATGGAGACACACAGCATCCTCAAAACGCTGGAAAGAAAAGATAGACAAAACGAGGAAGGAAATTGAGACAACACAGAATTACCTAATGGATATTAAGAACCGCATAATACCACCCTTTTTTAAATCTGAATTGAACCAGTTATATCAGTCTCAGTATAATCACCTGGACAGTCTTTCCAAAAAACTGCCTTCTTTTACAAAGAAAAATGAAGATGCAAACGAAACAGCTGTTCAAGATACATCTGATCTTAATCTAGATTTCAAGCCACATAAGGTTTCAGAACAGAAATCCTCAAGTGTGCCTAGTCTTCCAAAACCAGAGATTTCTGCAGATAAGAAGGATTTCACTGCTCAGAACCAAACTGAAAATTTAACAAAATCTCCTGAAGAAGCAGAATCTTATGAGCCCGTGAAAATTCCAACCCAGTCCTTGCTGCAGGATGTTGCGGGACAAACAAGAAAAGAAAAAGTGAAGTTGCCTCATTATTTGCTGAGTTCCAAGCCTAAGTCTCAACCTCTTGCAAAGGTGCAAGATTCTGTTGGAGGAAAAGTGAACACATCCTCTGTTGCATCTGCTGCCATTAATAATGCAAAGTCATCCCTTTTTGGGTTCCATCTTCTCCCATCATCAGTCAAGTTTGGAGTGCTTAAGGAAGGACATACCTATGCCACAGTTGTAAAGCTCAAGAATGTTGGAGTGGACTTCTGCAGGTTTAAAGTAAAGCAGCCCCCACCCAGCACAGGACTGAAAGTGACTTACAAACCTGGACCTGTGGCAGCTGGTATGCAGACAGAACTGAATATAGAGTTATTTGCCACAGCTGTTGGAGAGGATGGGGCCAAGGGATCAGCACACATCTCTCACAATATCGAGATTATGACAGAGCATGAGGTTCTGTTCCTACCTGTGGAAGCAACTGTTTTAACAAGCAGCAATTATGATAAACGACCAAAAGACTTTCCCCAGGGAAAAGAAAATCCAATGGTCCAGAGAACTTCTACAATTTATTCCTCCACACTTGGAGTCTTCATGTCTCGTAAAGTTTCTCCACATTAG

SEQ ID NO:2：野生型SPAG17蛋白的氨基酸序列

MAPKKEKGGTVNTSSKIWEPSLIAAQFNQNDWQASIAFVVGNQIEDDLLIQALTVAVQVPQRKLFSMVSWQDILQQINEINTLVGSASSKKAKKPVGGNAPLYYEVLTAAKAIMDSGEKLTLPLIGKLLKFQLLQIKFKDQQRRENEKKVIEDKPKLEKDKGKAKSPKEKKAPSAKPAKGKGKDQPEANAPVKKTTQLKRRGEDDHTNRYIDDEPDDGAQHYIIVVGFNNPQLLAIMAELGIPITSVIKISSENYEPLQTHLAAVNQQQEVLLQSEDLEAEKLKKENAIKELKTFWKYLEPVLNNEKPETNLFDVARLEYMVKAADFPSDWSDGEMMLKLGTDIFENIACLMYDILDWKRQHQHYLESMQLINVPQVVNEKPVLEAMPTSEAPQPAVPAPGKKKAQYEEPQAPPPVTSVITTEVDMRYYNYLLNPIREEFISVPLILHCMLEQVVATEEDLVPPSLREPSPRADGLDHRIAAHIVSLLPSLCLSEREKKNLHDIFLSEEENESKAVPKGPLLLNYHDAHAHKKYALQDQKNFDPVQIEQEMQSKLPLWEFLQFPLPPPWNNTKRLATIHELMHFCTSDVLSWNEVERAFKVFTFESLKLSEVDEKGKLKPSGMMCGSDSEMFNIPWDNPARFAKQIRQQYVMKMNTQEAKQKADIKIKDRTLFVDQNLSMSVQDNESNREPSDPSQCDANNMKHSDLNNLKLSVPDNRQLLEQESIMKAQPQHESLEQTTNNEIKDDAVTKADSHEKKPKKMMVEADLEDIKKTQQRSLMDWSFTEHFKPKVLLQVLQEAHKQYRCVDSYYHTQDNSLLLVFHNPMNRQRLHCEYWNIALHSNVGFRNYLELVAKSIQDWITKEEAIYQESKMNEKIIRTRAELELKSSANAKLTSASKIFSIKESKSNKGISKTEISDQEKEKEKEKIPFILEGSLKAWKEEQHRLAEEERLREEKKAEKKGKEAGKKKGKDNAEKEDSRSLKKKSPYKEKSKEEQVKIQEVTEESPHQPEPKITYPFHGYNMGNIPTQISGSNYYLYPSDGGQIEVEKTMFEKGPTFIKVRVVKDNHNFMIHLNDPKEIVKKEEKGDYYLEEEEEGDEEQSLETEVSDAKNKAFSKFGSFSATLENGICLSISYYGSNGMAPEDKDPDLETILNIPSALTPTVVPVIVTVPQSKAKGKIKGKEKPKESLKEEEHPKEEEKKEEEVEPEPVLQETLDVPTFQSLNVSCPSGLLLTFIGQESTGQYVIDEEPTWDIMVRQSYPQRVKHYEFYKTVMPPAEQEASRVITSQGTVVKYMLDGSTQILFADGAVSRSPNSGLICPPSEMPATPHSGDLMDSISQQKSETIPSEITNTKKGKSHKSQSSMAHKGEIHDPPPEAVQTVTPVEVHIGTWFTTTPEGNRIGTKGLERIADLTPLLSFQATDPVNGTVMTTREDKVVIVERKDGTRIVDHADGTRITTFYQVYEDQIILPDDQETTEGPRTVTRQVKCMRVESSRYATVIANCEDSSCCATFGDGTTIIAKPQGTYQVLPPNTGSLYIDKDCSAVYCHESSSNIYYPFQKREQLRAGRYIMRHTSEVICEVLDPEGNTFQVMADGSISTILPEKKLEDDLNEKTEGYDSLSSMHLEKNHQQIYGEHVPRFFVMYADGSGMELLRDSDIEEYLSLAYKESNTVVLQEPVQEQPGTLTITVLRPFHEASPWQVKKEDTIVPPNLRSRSWETFPSVEKKTPGPPFGTQIWKGLCIESKQLVSAPGAILKSPSVLQMRQFIQHEVIKNEVKLRLQVSLKDYINYILKKEDELQEMMVKDSRTEEERGNAADLLKLVMSFPKMEETTKSHVTEVAAHLTDLFKQSLATPPKCPPDTFGKDFFEKTWRHTASSKRWKEKIDKTRKEIETTQNYLMDIKNRIIPPFFKSELNQLYQSQYNHLDSLSKKLPSFTKKNEDANETAVQDTSDLNLDFKPHKVSEQKSSSVPSLPKPEISADKKDFTAQNQTENLTKSPEEAESYEPVKIPTQSLLQDVAGQTRKEKVKLPHYLLSSKPKSQPLAKVQDSVGGKVNTSSVASAAINNAKSSLFGFHLLPSSVKFGVLKEGHTYATVVKLKNVGVDFCRFKVKQPPPSTGLKVTYKPGPVAAGMQTELNIELFATAVGEDGAKGSAHISHNIEIMTEHEVLFLPVEATVLTSSNYDKRPKDFPQGKENPMVQRTSTIYSSTLGVFMSRKVSPH。

序列表

<110>厦门市妇幼保健院（厦门市计划生育服务中心），首都医科大学附属北京妇产医院，烟台毓璜顶医院

<120>一种严重弱精子症新致病基因及其应用

<130> 2017

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 6672

<212> DNA

<213> Homo Sapiens

<400> 1

atggcaccca agaaggagaa aggaggaact gtgaacacca gttctaagat atgggaaccc 60

tcgctcatag ctgcacagtt caatcagaac gattggcagg cctccattgc ttttgtggtt 120

gggaaccaga ttgaagatga tcttctcatc caagccctta ccgtggctgt ccaggtccct 180

cagcgtaaac tcttcagtat ggtgtcgtgg caagacattc tccagcagat taatgaaata 240

aatacacttg ttggatctgc ttcatctaaa aaggcaaaaa aacctgtagg tggtaatgct 300

cctttatatt atgaggtgtt aacggcagca aaagcaatta tggatagtgg agagaaatta 360

accttaccac tgatagggaa actcttgaaa tttcaacttc tccagattaa atttaaggac 420

caacagcgac gggaaaatga aaagaaggta atagaagaca aacctaagtt agaaaaggat 480

aaagggaaag caaaatctcc caaggagaaa aaggctccaa gtgccaagcc tgccaaagga 540

aagggaaagg atcagcctga ggcaaatgca ccagtgaaaa agaccaccca gttaaagcgg 600

agaggagaag acgaccacac caatcgttac attgacgatg agccagatga tggtgcccaa 660

cattacatta tagttgtggg ctttaacaat cctcagctat tagcaattat ggctgagctt 720

ggcattccta taaccagcgt gattaaaata tcttcagaga attatgaacc tctgcagaca 780

cacctggcag cagttaacca gcagcaggaa gttcttcttc agtcagaaga tctagaagca 840

gaaaaattga agaaagaaaa tgccataaaa gagcttaaaa ctttctggaa gtacttggaa 900

ccagtcctga ataatgagaa acctgaaaca aatctctttg atgttgctcg acttgagtac 960

atggtcaaag cagctgattt tccttctgac tggtcagatg gtgagatgat gctgaaattg 1020

ggcactgata tttttgaaaa tattgcctgc ttgatgtatg acatcctgga ttggaaaagg 1080

cagcaccagc actatttgga aagcatgcag cttattaatg ttccacaagt ggttaatgag 1140

aaacctgtat tagaagccat gccaacttca gaggctccac aacctgctgt accagctcct 1200

ggaaagaaga aagcacagta tgaagaaccg caagctccac caccagtgac ttcagtcatc 1260

acaactgaag tagacatgag atattacaat tatttgctga atccaattcg agaggaattc 1320

atttctgtgc ccctgatact gcattgtatg ctggaacagg ttgttgcaac tgaagaagat 1380

ctcgtcccac ccagtctgcg ggagccatcc cccagagcag acgggctaga ccacagaatc 1440

gcagctcaca ttgtgtccct tctgccctca ctctgtctct cagagaggga gaaaaagaat 1500

cttcatgaca tatttttatc tgaagaagaa aatgaaagca aagcagtgcc caaaggcccc 1560

ctcctactga actatcatga tgcacacgcc cacaagaagt acgcactaca ggaccaaaag 1620

aattttgatc cagttcaaat tgagcaggag atgcagtcca agttgccact gtgggaattt 1680

cttcaattcc ctctaccccc accatggaac aacactaaac gtctagctac aattcatgag 1740

cttatgcact tttgtacgag tgatgtcttg agctggaatg aagtagaacg agccttcaag 1800

gtgtttactt ttgagagcct gaagctctct gaggttgatg aaaaagggaa actgaaacct 1860

tctgggatga tgtgtgggtc agattctgaa atgttcaaca taccgtggga caaccctgcc 1920

agatttgcta aacagataag gcagcaatat gtcatgaaaa tgaatactca agaggccaag 1980

cagaaagcag atattaaaat caaagacaga acactatttg tggatcagaa tttgtcaatg 2040

tctgtgcaag ataatgaaag caaccgagaa ccttcagatc ctagtcagtg tgatgctaac 2100

aatatgaagc attctgactt gaataatctc aaactctcag tccctgataa tagacagctg 2160

ttagagcagg agagcatcat gaaggctcag ccccaacatg agtctctgga gcagaccaca 2220

aacaatgaga tcaaagatga tgcagtcaca aaggctgatt ctcatgaaaa gaaacccaag 2280

aagatgatgg tggaagcaga tttagaggac ataaagaaaa cacagcagcg cagtctaatg 2340

gactggagtt ttactgaaca ttttaaaccg aaagtactgc ttcaggtcct tcaagaagcc 2400

cataagcaat ataggtgtgt tgattcttac taccacaccc aagacaactc tttactttta 2460

gtctttcaca atccaatgaa tagacaacgt ttgcattgtg aatattggaa cattgctctc 2520

cactccaatg ttggattcag gaattatttg gaacttgttg caaaatctat tcaagattgg 2580

attacaaaag aagaagctat atatcaggaa tctaaaatga atgagaaaat catcaggacc 2640

agagctgagc tggaattgaa atcttctgct aatgccaaac ttacttctgc tagcaaaatt 2700

ttttccatta aagaatctaa aagtaacaaa ggaatcagca aaacagagat atcagatcaa 2760

gaaaaagaaa aagagaagga aaagattcct ttcattttag aaggctctct caaggcatgg 2820

aaagaagagc aacatcgatt agcagaagag gagcgcttaa gggaagaaaa gaaagcagag 2880

aagaagggta aagaagctgg taaaaagaaa ggcaaggata acgcagagaa agaggatagt 2940

aggtctttga agaaaaaatc accttacaag gagaaatcta aagaagaaca agtcaagatc 3000

caagaagtaa cagaagagtc cccccaccaa ccagaaccta agataactta cccgtttcac 3060

ggatacaata tgggaaatat acccactcaa atctcagggt caaattacta cctgtatcct 3120

tctgatgggg ggcagattga agtggaaaag acaatgtttg aaaaaggccc aacttttatc 3180

aaagtgagag tggtaaagga caaccacaat tttatgattc atttaaatga ccctaaggaa 3240

attgtgaaaa aggaagagaa aggggattat tatttagaag aggaagaaga aggagatgag 3300

gaacaaagtc ttgaaacgga agtatcagat gcaaagaata aagctttcag caagtttgga 3360

tctttttctg ccaccttaga aaatggaatc tgcctctcga taagttacta tggatcaaat 3420

ggaatggcac cagaagataa ggatcctgat ttagaaacaa tattgaatat cccttcagca 3480

ctcactccaa cagtggttcc tgttatagtg accgttcctc aaagcaaagc taaagggaaa 3540

ataaaaggca aagaaaaacc caaagaatcc cttaaagaag aagaacaccc aaaagaagaa 3600

gagaaaaagg aagaagaagt agaaccagaa cctgttttac aagagacttt ggatgttccc 3660

accttccaga gcctaaatgt gtcttgcccc agtgggctcc tgttgacttt cattggacaa 3720

gaatctacag gtcaatatgt tatagatgag gaacccacct gggacatcat ggtccgtcag 3780

agctaccccc agagggtgaa gcactatgag ttctataaaa cggtgatgcc acccgcagag 3840

caggaggctt caagggttat caccagtcaa ggcactgttg tcaaatatat gttggatgga 3900

tccacacaga ttctctttgc agatggtgct gtgagcagga gtcccaattc aggtcttatt 3960

tgtcctcctt ctgaaatgcc agcaacgcct cacagtggag atttgatgga ctctatttct 4020

cagcagaaat cagaaacgat accatctgag attaccaaca caaagaaagg aaaaagtcac 4080

aaaagtcagt catcaatggc ccataagggt gaaatccatg accctcctcc agaggcagtt 4140

caaactgtaa ctcctgtgga ggttcacata ggcacctggt ttacaaccac acctgaagga 4200

aatcggatcg gcaccaaagg attagaaaga atagcagact tgaccccatt gttatccttt 4260

caggccacag atcctgtcaa tggaacggtt atgacaactc gagaagacaa agttgtcata 4320

gttgaaagga aagatggtac tcggatagtg gatcatgctg atggtaccag aatcacaacc 4380

ttttatcaag tttatgaaga tcaaattatt ctgccagatg atcaagaaac aaccgagggt 4440

cctcggactg tcaccaggca ggtgaagtgt atgcgggtag aaagctcacg ctatgccact 4500

gttatcgcca actgtgagga cagtagctgc tgtgccacct ttggagatgg aacaactatt 4560

attgcaaagc cacagggaac ataccaggtg ttacctccaa acacaggctc tctttatatt 4620

gacaaggatt gttcagctgt gtactgccat gagtcaagca gtaatatata ctatcctttt 4680

caaaagcgtg agcagctgcg agctggcagg tacatcatga ggcatacttc agaggttatc 4740

tgtgaggttc tggatcctga gggaaacact tttcaggtca tggctgatgg tagcatatca 4800

actatattac ctgaaaaaaa attggaagat gatttaaatg agaaaactga gggctatgat 4860

agtctgtcct ctatgcacct tgaaaagaat catcagcaaa tctatggtga acatgtcccc 4920

aggttttttg ttatgtatgc tgatggatca ggaatggaac ttcttcgaga cagtgacata 4980

gaagaatatc tatctttggc atataaagaa tcaaatactg ttgttctcca agagccagtg 5040

caggaacagc caggcaccct aaccatcaca gtccttcgcc ctttccatga agcatcacca 5100

tggcaagtaa aaaaggaaga tacaattgtc cctcctaatc tccggtcaag gtcatgggaa 5160

acatttccct cagttgagaa aaaaactcca ggacctccgt ttggtactca gatttggaaa 5220

ggcctttgca ttgagtccaa acagctagtg agtgccccgg gtgccatact caagagcccc 5280

agtgtgctac agatgcgcca attcattcag catgaggtca taaagaatga ggtgaaactg 5340

aggctgcagg tttcccttaa ggattacata aactatattc taaagaaaga agatgagctg 5400

caggaaatga tggttaaaga ttccagaact gaggaggaga gaggcaatgc tgctgatctc 5460

ctcaagctgg ttatgtcttt ccctaaaatg gaggaaacta caaaaagtca tgttactgaa 5520

gttgcagctc acctaactga tttattcaag cagtctttgg ctacgcctcc aaaatgccca 5580

ccagacacat ttggtaaaga tttctttgaa aagacatgga gacacacagc atcctcaaaa 5640

cgctggaaag aaaagataga caaaacgagg aaggaaattg agacaacaca gaattaccta 5700

atggatatta agaaccgcat aataccaccc ttttttaaat ctgaattgaa ccagttatat 5760

cagtctcagt ataatcacct ggacagtctt tccaaaaaac tgccttcttt tacaaagaaa 5820

aatgaagatg caaacgaaac agctgttcaa gatacatctg atcttaatct agatttcaag 5880

ccacataagg tttcagaaca gaaatcctca agtgtgccta gtcttccaaa accagagatt 5940

tctgcagata agaaggattt cactgctcag aaccaaactg aaaatttaac aaaatctcct 6000

gaagaagcag aatcttatga gcccgtgaaa attccaaccc agtccttgct gcaggatgtt 6060

gcgggacaaa caagaaaaga aaaagtgaag ttgcctcatt atttgctgag ttccaagcct 6120

aagtctcaac ctcttgcaaa ggtgcaagat tctgttggag gaaaagtgaa cacatcctct 6180

gttgcatctg ctgccattaa taatgcaaag tcatcccttt ttgggttcca tcttctccca 6240

tcatcagtca agtttggagt gcttaaggaa ggacatacct atgccacagt tgtaaagctc 6300

aagaatgttg gagtggactt ctgcaggttt aaagtaaagc agcccccacc cagcacagga 6360

ctgaaagtga cttacaaacc tggacctgtg gcagctggta tgcagacaga actgaatata 6420

gagttatttg ccacagctgt tggagaggat ggggccaagg gatcagcaca catctctcac 6480

aatatcgaga ttatgacaga gcatgaggtt ctgttcctac ctgtggaagc aactgtttta 6540

acaagcagca attatgataa acgaccaaaa gactttcccc agggaaaaga aaatccaatg 6600

gtccagagaa cttctacaat ttattcctcc acacttggag tcttcatgtc tcgtaaagtt 6660

tctccacatt ag 6672

<210> 2

<211> 2223

<212> PRT

<213> Homo Sapiens

<400> 2

Met Ala Pro Lys Lys Glu Lys Gly Gly Thr Val Asn Thr Ser Ser Lys

1 5 10 15

Ile Trp Glu Pro Ser Leu Ile Ala Ala Gln Phe Asn Gln Asn Asp Trp

20 25 30

Gln Ala Ser Ile Ala Phe Val Val Gly Asn Gln Ile Glu Asp Asp Leu

35 40 45

Leu Ile Gln Ala Leu Thr Val Ala Val Gln Val Pro Gln Arg Lys Leu

50 55 60

Phe Ser Met Val Ser Trp Gln Asp Ile Leu Gln Gln Ile Asn Glu Ile

65 70 75 80

Asn Thr Leu Val Gly Ser Ala Ser Ser Lys Lys Ala Lys Lys Pro Val

85 90 95

Gly Gly Asn Ala Pro Leu Tyr Tyr Glu Val Leu Thr Ala Ala Lys Ala

100 105 110

Ile Met Asp Ser Gly Glu Lys Leu Thr Leu Pro Leu Ile Gly Lys Leu

115 120 125

Leu Lys Phe Gln Leu Leu Gln Ile Lys Phe Lys Asp Gln Gln Arg Arg

130 135 140

Glu Asn Glu Lys Lys Val Ile Glu Asp Lys Pro Lys Leu Glu Lys Asp

145 150 155 160

Lys Gly Lys Ala Lys Ser Pro Lys Glu Lys Lys Ala Pro Ser Ala Lys

165 170 175

Pro Ala Lys Gly Lys Gly Lys Asp Gln Pro Glu Ala Asn Ala Pro Val

180 185 190

Lys Lys Thr Thr Gln Leu Lys Arg Arg Gly Glu Asp Asp His Thr Asn

195 200 205

Arg Tyr Ile Asp Asp Glu Pro Asp Asp Gly Ala Gln His Tyr Ile Ile

210 215 220

Val Val Gly Phe Asn Asn Pro Gln Leu Leu Ala Ile Met Ala Glu Leu

225 230 235 240

Gly Ile Pro Ile Thr Ser Val Ile Lys Ile Ser Ser Glu Asn Tyr Glu

245 250 255

Pro Leu Gln Thr His Leu Ala Ala Val Asn Gln Gln Gln Glu Val Leu

260 265 270

Leu Gln Ser Glu Asp Leu Glu Ala Glu Lys Leu Lys Lys Glu Asn Ala

275 280 285

Ile Lys Glu Leu Lys Thr Phe Trp Lys Tyr Leu Glu Pro Val Leu Asn

290 295 300

Asn Glu Lys Pro Glu Thr Asn Leu Phe Asp Val Ala Arg Leu Glu Tyr

305 310 315 320

Met Val Lys Ala Ala Asp Phe Pro Ser Asp Trp Ser Asp Gly Glu Met

325 330 335

Met Leu Lys Leu Gly Thr Asp Ile Phe Glu Asn Ile Ala Cys Leu Met

340 345 350

Tyr Asp Ile Leu Asp Trp Lys Arg Gln His Gln His Tyr Leu Glu Ser

355 360 365

Met Gln Leu Ile Asn Val Pro Gln Val Val Asn Glu Lys Pro Val Leu

370 375 380

Glu Ala Met Pro Thr Ser Glu Ala Pro Gln Pro Ala Val Pro Ala Pro

385 390 395 400

Gly Lys Lys Lys Ala Gln Tyr Glu Glu Pro Gln Ala Pro Pro Pro Val

405 410 415

Thr Ser Val Ile Thr Thr Glu Val Asp Met Arg Tyr Tyr Asn Tyr Leu

420 425 430

Leu Asn Pro Ile Arg Glu Glu Phe Ile Ser Val Pro Leu Ile Leu His

435 440 445

Cys Met Leu Glu Gln Val Val Ala Thr Glu Glu Asp Leu Val Pro Pro

450 455 460

Ser Leu Arg Glu Pro Ser Pro Arg Ala Asp Gly Leu Asp His Arg Ile

465 470 475 480

Ala Ala His Ile Val Ser Leu Leu Pro Ser Leu Cys Leu Ser Glu Arg

485 490 495

Glu Lys Lys Asn Leu His Asp Ile Phe Leu Ser Glu Glu Glu Asn Glu

500 505 510

Ser Lys Ala Val Pro Lys Gly Pro Leu Leu Leu Asn Tyr His Asp Ala

515 520 525

His Ala His Lys Lys Tyr Ala Leu Gln Asp Gln Lys Asn Phe Asp Pro

530 535 540

Val Gln Ile Glu Gln Glu Met Gln Ser Lys Leu Pro Leu Trp Glu Phe

545 550 555 560

Leu Gln Phe Pro Leu Pro Pro Pro Trp Asn Asn Thr Lys Arg Leu Ala

565 570 575

Thr Ile His Glu Leu Met His Phe Cys Thr Ser Asp Val Leu Ser Trp

580 585 590

Asn Glu Val Glu Arg Ala Phe Lys Val Phe Thr Phe Glu Ser Leu Lys

595 600 605

Leu Ser Glu Val Asp Glu Lys Gly Lys Leu Lys Pro Ser Gly Met Met

610 615 620

Cys Gly Ser Asp Ser Glu Met Phe Asn Ile Pro Trp Asp Asn Pro Ala

625 630 635 640

Arg Phe Ala Lys Gln Ile Arg Gln Gln Tyr Val Met Lys Met Asn Thr

645 650 655

Gln Glu Ala Lys Gln Lys Ala Asp Ile Lys Ile Lys Asp Arg Thr Leu

660 665 670

Phe Val Asp Gln Asn Leu Ser Met Ser Val Gln Asp Asn Glu Ser Asn

675 680 685

Arg Glu Pro Ser Asp Pro Ser Gln Cys Asp Ala Asn Asn Met Lys His

690 695 700

Ser Asp Leu Asn Asn Leu Lys Leu Ser Val Pro Asp Asn Arg Gln Leu

705 710 715 720

Leu Glu Gln Glu Ser Ile Met Lys Ala Gln Pro Gln His Glu Ser Leu

725 730 735

Glu Gln Thr Thr Asn Asn Glu Ile Lys Asp Asp Ala Val Thr Lys Ala

740 745 750

Asp Ser His Glu Lys Lys Pro Lys Lys Met Met Val Glu Ala Asp Leu

755 760 765

Glu Asp Ile Lys Lys Thr Gln Gln Arg Ser Leu Met Asp Trp Ser Phe

770 775 780

Thr Glu His Phe Lys Pro Lys Val Leu Leu Gln Val Leu Gln Glu Ala

785 790 795 800

His Lys Gln Tyr Arg Cys Val Asp Ser Tyr Tyr His Thr Gln Asp Asn

805 810 815

Ser Leu Leu Leu Val Phe His Asn Pro Met Asn Arg Gln Arg Leu His

820 825 830

Cys Glu Tyr Trp Asn Ile Ala Leu His Ser Asn Val Gly Phe Arg Asn

835 840 845

Tyr Leu Glu Leu Val Ala Lys Ser Ile Gln Asp Trp Ile Thr Lys Glu

850 855 860

Glu Ala Ile Tyr Gln Glu Ser Lys Met Asn Glu Lys Ile Ile Arg Thr

865 870 875 880

Arg Ala Glu Leu Glu Leu Lys Ser Ser Ala Asn Ala Lys Leu Thr Ser

885 890 895

Ala Ser Lys Ile Phe Ser Ile Lys Glu Ser Lys Ser Asn Lys Gly Ile

900 905 910

Ser Lys Thr Glu Ile Ser Asp Gln Glu Lys Glu Lys Glu Lys Glu Lys

915 920 925

Ile Pro Phe Ile Leu Glu Gly Ser Leu Lys Ala Trp Lys Glu Glu Gln

930 935 940

His Arg Leu Ala Glu Glu Glu Arg Leu Arg Glu Glu Lys Lys Ala Glu

945 950 955 960

Lys Lys Gly Lys Glu Ala Gly Lys Lys Lys Gly Lys Asp Asn Ala Glu

965 970 975

Lys Glu Asp Ser Arg Ser Leu Lys Lys Lys Ser Pro Tyr Lys Glu Lys

980 985 990

Ser Lys Glu Glu Gln Val Lys Ile Gln Glu Val Thr Glu Glu Ser Pro

995 1000 1005

His Gln Pro Glu Pro Lys Ile Thr Tyr Pro Phe His Gly Tyr Asn

1010 1015 1020

Met Gly Asn Ile Pro Thr Gln Ile Ser Gly Ser Asn Tyr Tyr Leu

1025 1030 1035

Tyr Pro Ser Asp Gly Gly Gln Ile Glu Val Glu Lys Thr Met Phe

1040 1045 1050

Glu Lys Gly Pro Thr Phe Ile Lys Val Arg Val Val Lys Asp Asn

1055 1060 1065

His Asn Phe Met Ile His Leu Asn Asp Pro Lys Glu Ile Val Lys

1070 1075 1080

Lys Glu Glu Lys Gly Asp Tyr Tyr Leu Glu Glu Glu Glu Glu Gly

1085 1090 1095

Asp Glu Glu Gln Ser Leu Glu Thr Glu Val Ser Asp Ala Lys Asn

1100 1105 1110

Lys Ala Phe Ser Lys Phe Gly Ser Phe Ser Ala Thr Leu Glu Asn

1115 1120 1125

Gly Ile Cys Leu Ser Ile Ser Tyr Tyr Gly Ser Asn Gly Met Ala

1130 1135 1140

Pro Glu Asp Lys Asp Pro Asp Leu Glu Thr Ile Leu Asn Ile Pro

1145 1150 1155

Ser Ala Leu Thr Pro Thr Val Val Pro Val Ile Val Thr Val Pro

1160 1165 1170

Gln Ser Lys Ala Lys Gly Lys Ile Lys Gly Lys Glu Lys Pro Lys

1175 1180 1185

Glu Ser Leu Lys Glu Glu Glu His Pro Lys Glu Glu Glu Lys Lys

1190 1195 1200

Glu Glu Glu Val Glu Pro Glu Pro Val Leu Gln Glu Thr Leu Asp

1205 1210 1215

Val Pro Thr Phe Gln Ser Leu Asn Val Ser Cys Pro Ser Gly Leu

1220 1225 1230

Leu Leu Thr Phe Ile Gly Gln Glu Ser Thr Gly Gln Tyr Val Ile

1235 1240 1245

Asp Glu Glu Pro Thr Trp Asp Ile Met Val Arg Gln Ser Tyr Pro

1250 1255 1260

Gln Arg Val Lys His Tyr Glu Phe Tyr Lys Thr Val Met Pro Pro

1265 1270 1275

Ala Glu Gln Glu Ala Ser Arg Val Ile Thr Ser Gln Gly Thr Val

1280 1285 1290

Val Lys Tyr Met Leu Asp Gly Ser Thr Gln Ile Leu Phe Ala Asp

1295 1300 1305

Gly Ala Val Ser Arg Ser Pro Asn Ser Gly Leu Ile Cys Pro Pro

1310 1315 1320

Ser Glu Met Pro Ala Thr Pro His Ser Gly Asp Leu Met Asp Ser

1325 1330 1335

Ile Ser Gln Gln Lys Ser Glu Thr Ile Pro Ser Glu Ile Thr Asn

1340 1345 1350

Thr Lys Lys Gly Lys Ser His Lys Ser Gln Ser Ser Met Ala His

1355 1360 1365

Lys Gly Glu Ile His Asp Pro Pro Pro Glu Ala Val Gln Thr Val

1370 1375 1380

Thr Pro Val Glu Val His Ile Gly Thr Trp Phe Thr Thr Thr Pro

1385 1390 1395

Glu Gly Asn Arg Ile Gly Thr Lys Gly Leu Glu Arg Ile Ala Asp

1400 1405 1410

Leu Thr Pro Leu Leu Ser Phe Gln Ala Thr Asp Pro Val Asn Gly

1415 1420 1425

Thr Val Met Thr Thr Arg Glu Asp Lys Val Val Ile Val Glu Arg

1430 1435 1440

Lys Asp Gly Thr Arg Ile Val Asp His Ala Asp Gly Thr Arg Ile

1445 1450 1455

Thr Thr Phe Tyr Gln Val Tyr Glu Asp Gln Ile Ile Leu Pro Asp

1460 1465 1470

Asp Gln Glu Thr Thr Glu Gly Pro Arg Thr Val Thr Arg Gln Val

1475 1480 1485

Lys Cys Met Arg Val Glu Ser Ser Arg Tyr Ala Thr Val Ile Ala

1490 1495 1500

Asn Cys Glu Asp Ser Ser Cys Cys Ala Thr Phe Gly Asp Gly Thr

1505 1510 1515

Thr Ile Ile Ala Lys Pro Gln Gly Thr Tyr Gln Val Leu Pro Pro

1520 1525 1530

Asn Thr Gly Ser Leu Tyr Ile Asp Lys Asp Cys Ser Ala Val Tyr

1535 1540 1545

Cys His Glu Ser Ser Ser Asn Ile Tyr Tyr Pro Phe Gln Lys Arg

1550 1555 1560

Glu Gln Leu Arg Ala Gly Arg Tyr Ile Met Arg His Thr Ser Glu

1565 1570 1575

Val Ile Cys Glu Val Leu Asp Pro Glu Gly Asn Thr Phe Gln Val

1580 1585 1590

Met Ala Asp Gly Ser Ile Ser Thr Ile Leu Pro Glu Lys Lys Leu

1595 1600 1605

Glu Asp Asp Leu Asn Glu Lys Thr Glu Gly Tyr Asp Ser Leu Ser

1610 1615 1620

Ser Met His Leu Glu Lys Asn His Gln Gln Ile Tyr Gly Glu His

1625 1630 1635

Val Pro Arg Phe Phe Val Met Tyr Ala Asp Gly Ser Gly Met Glu

1640 1645 1650

Leu Leu Arg Asp Ser Asp Ile Glu Glu Tyr Leu Ser Leu Ala Tyr

1655 1660 1665

Lys Glu Ser Asn Thr Val Val Leu Gln Glu Pro Val Gln Glu Gln

1670 1675 1680

Pro Gly Thr Leu Thr Ile Thr Val Leu Arg Pro Phe His Glu Ala

1685 1690 1695

Ser Pro Trp Gln Val Lys Lys Glu Asp Thr Ile Val Pro Pro Asn

1700 1705 1710

Leu Arg Ser Arg Ser Trp Glu Thr Phe Pro Ser Val Glu Lys Lys

1715 1720 1725

Thr Pro Gly Pro Pro Phe Gly Thr Gln Ile Trp Lys Gly Leu Cys

1730 1735 1740

Ile Glu Ser Lys Gln Leu Val Ser Ala Pro Gly Ala Ile Leu Lys

1745 1750 1755

Ser Pro Ser Val Leu Gln Met Arg Gln Phe Ile Gln His Glu Val

1760 1765 1770

Ile Lys Asn Glu Val Lys Leu Arg Leu Gln Val Ser Leu Lys Asp

1775 1780 1785

Tyr Ile Asn Tyr Ile Leu Lys Lys Glu Asp Glu Leu Gln Glu Met

1790 1795 1800

Met Val Lys Asp Ser Arg Thr Glu Glu Glu Arg Gly Asn Ala Ala

1805 1810 1815

Asp Leu Leu Lys Leu Val Met Ser Phe Pro Lys Met Glu Glu Thr

1820 1825 1830

Thr Lys Ser His Val Thr Glu Val Ala Ala His Leu Thr Asp Leu

1835 1840 1845

Phe Lys Gln Ser Leu Ala Thr Pro Pro Lys Cys Pro Pro Asp Thr

1850 1855 1860

Phe Gly Lys Asp Phe Phe Glu Lys Thr Trp Arg His Thr Ala Ser

1865 1870 1875

Ser Lys Arg Trp Lys Glu Lys Ile Asp Lys Thr Arg Lys Glu Ile

1880 1885 1890

Glu Thr Thr Gln Asn Tyr Leu Met Asp Ile Lys Asn Arg Ile Ile

1895 1900 1905

Pro Pro Phe Phe Lys Ser Glu Leu Asn Gln Leu Tyr Gln Ser Gln

1910 1915 1920

Tyr Asn His Leu Asp Ser Leu Ser Lys Lys Leu Pro Ser Phe Thr

1925 1930 1935

Lys Lys Asn Glu Asp Ala Asn Glu Thr Ala Val Gln Asp Thr Ser

1940 1945 1950

Asp Leu Asn Leu Asp Phe Lys Pro His Lys Val Ser Glu Gln Lys

1955 1960 1965

Ser Ser Ser Val Pro Ser Leu Pro Lys Pro Glu Ile Ser Ala Asp

1970 1975 1980

Lys Lys Asp Phe Thr Ala Gln Asn Gln Thr Glu Asn Leu Thr Lys

1985 1990 1995

Ser Pro Glu Glu Ala Glu Ser Tyr Glu Pro Val Lys Ile Pro Thr

2000 2005 2010

Gln Ser Leu Leu Gln Asp Val Ala Gly Gln Thr Arg Lys Glu Lys

2015 2020 2025

Val Lys Leu Pro His Tyr Leu Leu Ser Ser Lys Pro Lys Ser Gln

2030 2035 2040

Pro Leu Ala Lys Val Gln Asp Ser Val Gly Gly Lys Val Asn Thr

2045 2050 2055

Ser Ser Val Ala Ser Ala Ala Ile Asn Asn Ala Lys Ser Ser Leu

2060 2065 2070

Phe Gly Phe His Leu Leu Pro Ser Ser Val Lys Phe Gly Val Leu

2075 2080 2085

Lys Glu Gly His Thr Tyr Ala Thr Val Val Lys Leu Lys Asn Val

2090 2095 2100

Gly Val Asp Phe Cys Arg Phe Lys Val Lys Gln Pro Pro Pro Ser

2105 2110 2115

Thr Gly Leu Lys Val Thr Tyr Lys Pro Gly Pro Val Ala Ala Gly

2120 2125 2130

Met Gln Thr Glu Leu Asn Ile Glu Leu Phe Ala Thr Ala Val Gly

2135 2140 2145

Glu Asp Gly Ala Lys Gly Ser Ala His Ile Ser His Asn Ile Glu

2150 2155 2160

Ile Met Thr Glu His Glu Val Leu Phe Leu Pro Val Glu Ala Thr

2165 2170 2175

Val Leu Thr Ser Ser Asn Tyr Asp Lys Arg Pro Lys Asp Phe Pro

2180 2185 2190

Gln Gly Lys Glu Asn Pro Met Val Gln Arg Thr Ser Thr Ile Tyr

2195 2200 2205

Ser Ser Thr Leu Gly Val Phe Met Ser Arg Lys Val Ser Pro His

2210 2215 2220

<210> 3

<211> 20

<212> DNA

<213>人工序列

<400> 3

tggtgccatc ttcgtattca 20

<210> 4

<211> 20

<212> DNA

<213>人工序列

<400> 4

atagaccctg ctttggatgg 20

Claims

1.通过外显子组测序的方法确定严重弱精子症的新致病基因。

2.严重弱精子症的生物标记物，其特征在于即突变的SPAG17基因或SPAG17蛋白，所述生物标记物是具有选自如下的突变的SPAG17基因或SPAG17蛋白：

在外显子30中错义突变(c.4343G>A；p.R1448Q)。

3.突变的SPAG17基因，其特征在于为SEQ ID NO:1的序列中具有以下突变：

在外显子30中错义突变(c.4343G>A)；

突变的SPAG17蛋白，为SEQ ID NO:2的序列中具有以下突变：

在外显子30中错义突变(p.R1448Q)。

4.一种检测严重弱精子症的方法，其特征在于包括检测受试者的SPAG17基因或SPAG17蛋白中是否存在突变位点，若有突变位点，则所述受试者被鉴定为患有严重弱精子症或易患严重弱精子症，所述突变位点选自如下一种：

在外显子30中错义突变(c.4343G>A；p.R1448Q)；

所述SPAG17蛋白为SEQ ID NO:2的序列表示；

所述SPAG17基因为SEQ ID NO:1的序列表示。

5.检测严重弱精子症的方法，其特征在于包括如下至少一组引物扩增的步骤：

SEQ ID NO:3和SEQ ID NO:4。

6.检测严重弱精子症的方法，其特征在于检测突变位点通过选自如下进行：测序、电泳、核酸杂交、原位杂交、PCR、逆转录酶链反应和变性高效液相色谱。

7.通过PCR检测SPAG17基因或SPAG17蛋白突变中使用的引物对，其特征在于所述突变是选自如下一种：

在外显子30中错义突变(c.4343G>A；p.R1448Q)，

其中所述引物对分别基于选自如下的位置前后设计，使得扩增该位置，编号基于SPAG17的cDNA序列：4343。

8.与突变SPAG17基因互补的核酸探针，其特征在于所述突变是选自如下一种：

在外显子30中错义突变(c.4343G>A；p.R1448Q)，

所述探针与突变SPAG17基因的互补区包括选自如下的位置，编号基于SPAG17的cDNA序列：4343。

9.检测突变SPAG17基因或SPAG17蛋白的试剂盒，其特征在于包含一组或多组引物对，其中所述突变是选自如下一种：

在外显子30中错义突变(c.4343G>A；p.R1448Q)，

其中所述引物对分别基于选自如下的位置前后设计，使得其扩增产物涵盖该位置，编号基于SPAG17的cDNA序列：4343。

10.检测突变SPAG17基因或SPAG17蛋白的试剂盒，其特征在于包含选自如下的至少一组引物：

SEQ ID NO:3和SEQ ID NO:4。

11.检测突变SPAG17基因的试剂盒，其特征在于包含一个或多个核酸探针，所述突变是选自如下一种：

在外显子30中错义突变(c.4343G>A；p.R1448Q)，

所述探针与突变SPAG17基因上包含选自如下的位置的区域互补，编号基于SPAG17的cDNA序列：4343。

12.一种严重弱精子症新致病基因的应用。