CN112359047B - 一种突变PIKfyve基因及其应用 - Google Patents
一种突变PIKfyve基因及其应用 Download PDFInfo
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Abstract
本发明公开了一种突变的PIKfyve基因，其与人类野生型PIKfyve基因存在一个突变的差异，突变位于第2条染色体上的第39号外显子中c.5828G‑A，突变的基因序列为SEQ ID NO.1。本发明还公开了突变的PIKfyve基因的应用。本发明是一种先天性白内障的致病基因，该基因准确定位基因突变位点，为先天性白内障的检测和确认提供依据。
Description
技术领域
本发明涉及分子遗传学技术领域，具体涉及一种突变PIKfyve基因及其应用。
背景技术
先天性白内障(congenital cataract，CC)是指出生前后即存在或出生后一年内逐渐形成的先天遗传或发育障碍导致的白内障。是一组由于晶状体混浊且通常发生在出生时或儿童早期的疾病，是一种常见的视觉障碍性眼病，发病率约0.01％～0.06％，是造成儿童失明和弱视的重要原因，约占新生儿盲的30％，占中国儿童失明原因的第二位。先天性白内障可以是家族性或散发性，晶状体可表现有多种不同的混浊类型，如板层状、核性、粉尘状、蓝点状、花冠状、极性等；临床上可表现为单眼或双眼视力下降，症状严重者可有视野缺损、单眼复视、多视或眩光等，可伴有小角膜、小眼球及虹膜缺损等其他眼部异常或全身其他先天性异常。先天性白内障的病因可分为三大类：遗传因素、环境因素以及原因不明。约1/3先天性白内障的发生与遗传相关，其遗传方式主要分3种：常染色体显性遗传、常染色体隐性遗传及X连锁遗传，其中以常染色体显性遗传先天性白内障最多见。迄今超过30个基因的突变可导致单纯性先天性白内障。根据白内障致病基因的功能不同可分为晶状体蛋白基因(CRYA、CRYB、CRYG)、缝隙连接蛋白基因(GJA1、GJA3、GJA8)、膜蛋白基因(MIP、LIM2)、细胞骨架蛋白基因(BFSP2、EPHA2)、转录因子调节基因(HSF4、MAF、PITX3、PAX6)及其他基因。
目前，仍有许多先天性白内障相关的致病基因尚未发现或定位不明确，对此还需深入和广泛的研究，并需建立一种快速、简便的检测方法。
发明内容
针对现有技术的上述缺陷，本发明的目的在于提供一种突变PIKfyve基因，是一种先天性白内障的致病基因，该基因准确定位基因突变位点，为先天性白内障的检测和确认提供依据。
为实现上述目的，本发明采用的技术方案如下
一种突变的PIKfyve基因，其与人类野生型PIKfyve基因存在一个突变的差异，突变位于第2条染色体上的第39号外显子中c.5828G-A，突变的基因序列为SEQ ID NO.1。
本发明还提供了一种载体，该载体包含上述突变的PIKfyve基因。
本发明还提供了一种宿主细胞，该宿主细胞包含上述突变的PIKfyve基因，或包含上述载体。
本发明还提供了所述的突变的PIKfyve基因在制备CC动物模型、诊断试剂、检测/诊断试剂盒中的应用。
本发明还提供了所述的载体在制备CC动物模型、检测/诊断试剂盒中的应用。
本发明还提供了所述的宿主细胞在制备CC动物模型、检测/诊断试剂盒中的应用。
本发明还提供了一种用于诊断先天性白内障的诊断试剂，该诊断试剂包括能够特异性检测本发明所述突变的PIKfyve基因的引物或探针。
一种检测试剂盒，包含上述诊断试剂。
与现有技术相比，本发明的有益效果是：
1.本发明所述的突变的PIKfyve基因是一种先天性白内障的致病基因，其与人类野生型PIKfyve基因存在一个突变的差异，突变位于第2条染色体上的第39号外显子中c.5828G-A，PIKfyve基因序列变异的位点可用于检测人体生物样本中是否存在所述基因点突变的方法，可实现对一个先天性白内障家系的所有成员进行检测。
2.本发明所述的突变的PIKfyve基因应用在检测试剂盒中，可以精确地诊断PIKfyve基因突变所致的先天性白内障，为先天性白内障的诊断提供新的理论依据。
3.本发明所述的突变的PIKfyve基因所获得的疾病动物模型为研究先天性白内障的发病机制和治疗方法的提供了有力的实验工具。
附图说明
图1为先天性白内障家系系谱图(I-IV代表家系中的第一代至第四代)。
图2为先天性白内障家系中患病者III-9的左眼裂隙灯下弥散光照射法示晶状体中央区核性粉尘状混浊。
图3为PIKfyve基因Sanger法测序峰图对比图，其中A：表型正常者测序峰图，红色箭头示无突变；B：患病者测序峰图，红色箭头示c.G5828A。
图4为构建的斑马鱼模型，其中A为PIKfyve突变的斑马鱼，B为共聚焦下突变型斑马鱼的混浊晶状体(白内障)。
具体实施方式
下面，结合具体实施方式，对本发明做进一步描述：
在本发明中，若非特指，所有的份、百分比均为重量单位，所采用的设备和原料等均可从市场购得或是本领域常用的。下述实施例中的方法，如无特别说明，均为本领域的常规方法。
一种突变的PIKfyve基因，其与人类野生型PIKfyve基因存在一个突变的差异，突变位于第2条染色体上的第39号外显子中c.5828G-A，突变的基因序列为SEQ ID NO.1。在本发明中，SEQ ID NO.1是DNA序列，但是还可以包含与SEQ ID NO.1序列互补的序列，也可以包括RNA形式。SEQ ID NO.1是第39号外显子中的一段序列，突变位置位于SEQ ID NO.1中的第406个碱基位置，由原来G突变为A。
在本发明中，突变位于PIKfyve基因第2条染色体上的第39号外显子，第5828号碱基上的鸟嘌呤突变为腺嘌呤(c.G5828A)，导致对应编码序列上的甘氨酸突变为谷氨酸(p.G1943E)。
本发明中所述的“突变”可以指基因中某一个或某几个碱基的缺失或替换，或者是增加某一个或者某几个碱基。在本发明中突变的PIKfyve基因是通过其中的一个碱基被替换实现的突变。
本发明还提供了一种载体，该载体包含上述突变的PIKfyve基因。“载体”是可将突变的PIKfyve基因插入其中的一种核酸运载工具，载体可以通过转化、转染等方式转入宿主细胞，并可随宿主细胞分裂而传给后代。通用的载体可以但不限于质粒、噬菌体、动植物病毒等。
本发明还提供了一种宿主细胞，该宿主细胞包含上述突变的PIKfyve基因，或包含上述载体。
本发明还提供了所述的突变的PIKfyve基因在制备CC动物模型、诊断试剂、检测/诊断试剂盒中的应用。
本发明还提供了所述的载体在制备CC动物模型、检测/诊断试剂盒中的应用。
本发明还提供了所述的宿主细胞在制备CC动物模型、检测/诊断试剂盒中的应用。
本发明还提供了一种用于诊断先天性白内障的诊断试剂，该诊断试剂包括能够特异性检测本发明所述突变的PIKfyve基因的引物或探针。该方案中所述的能够特异性检测本发明所述突变的PIKfyve基因的探针或引物是设计的探针或引物能特异性与突变的PIKfyve基因结合，而不与其他基因结合，结合的方式可以但不限于杂交。在本发明中探针或引物的设计是本领域熟知的技术。
以下是本发明具体的实施例，以下实施例只是说明本发明能够实施的具体方案之一，不能用于限定本发明的保护范围。
实施例1
本发明一个先天性白内障家系，用全外显子测序及Sanger法测序对致病基因进行了染色体定位及候选基因突变检测。经过全外显子测序，筛选出4个候选致病基因(PIKfyve、NPHS1、FPR1、EPG5)。经过对所有家系成员Sanger法测序验证及家系共分离分析，最终明确了该家系的致病基因为突变的PIKfyve。
1)家系收集，采集了一个来自中国的先天性白内障家系，对所有成员进行了询问并记录成员病史、家族史及婚育史等，进行全面体格检查，明确是否伴有其他的眼部或全身系统相关合并症，0.5％托吡卡胺滴眼液散瞳后行眼前段图像数码采集。该家系共4代31名成员，其中12例患病者，男性9例，女性3例(参见图1)。患病者多在幼年时出现双眼视力低下，部分患者检查发现晶状体混浊(先证者临床表现如图2所示)；该家系中患病者未见其他眼部组织和全身异常情况。
2)致病基因的定位
a.血样采集及基因组DNA的提取：发明人采集该家系中21名成员的外周静脉血或带有毛囊的头发，其中包括患病者10例和表型正常的家系成员11例。将采集到的样本在-80℃温度条件下保存，用于基因组DNA的提取。采用DNA提取试剂盒，严格按照说明书提取基因组DNA。采用Qubit 2.0荧光定量仪测量DNA样本的浓度，计算其A260/A280nm值以评估DNA样本纯度；DNA终浓度调整为50ng/μL；取5μL于1.2％琼脂糖凝胶中电泳进行DNA样本的定性分析。将提取的基因组DNA及剩余血液样品置于-80℃冰箱保存备用；
b.全外显子组测序：发明人对家系中3个患病者(图1中II-1、III-9及IV-5)和1个正常者(图1中II-5)的外周血DNA进行全外显子组测序，筛选出可疑致病基因。具体步骤如下：将基因组DNA经Covaris S220破碎仪随机打断成长度为180-280bp的片段，采用AgilentSureSelect Human All Exon V5/V6试剂盒进行末端修复和加A尾后，在片段两端分别连接上接头制备DNA文库。带有特异性标记的文库经多重测序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线性扩增后进行文库质量检测。文库构建完成后，运用Illumina HiSeq 4000平台进行测序。原始测序数据经过数据质控等过滤处理(去除含有带接头信息或低质量的测序序列等)及错误率检查后得到有效序列；通过BWA软件比对到人类参考基因组(GRCh37/hg19)，经SAMtools软件对结果进行排序以及Picard软件标记重复测序数据，最后对采集的数据进行标准信息分析流程处理。通过ANNOVAR软件对SNP和InDel数据进行注释，参考数据库有dbSNP147、HGMD(human genemutation database)、NCBI ClinVar、GWAS(genome-wide association study)和ExAc(exome aggregation consortium)等。过滤标准包括：A.过滤千人基因组数据库，去除个体间多样性位点，保留1000G中频率低于0.01的变异位点，得到真正可能致病的罕见突变；B.保留外显子区或可变剪接位点区(splicing，上下10bp)的变异；C.保留非同义突变；D.依据SIFT、Polyphen、MutationTaster及CADD(combined annotation dependent depletion)4个软件的打分预测情况进行变异位点筛选，保留至少两个有分值的软件支持其可能有害的位点；E.遗传模式符合常染色体显性遗传规律。
c.Sanger测序
将采集到的所有家系成员DNA样品，针对全外显子组测序筛选出的可疑基因进行Sanger法测序验证，判断否符合表型-基因型共分离结果。通过Primer 5.0设计候选基因的上下游引物，再针对所有DNA样本对应的片段进行PCR扩增。应用PCR扩增仪扩增得到的产物在ABI 3730测序仪上进行Sanger法测序及分析。
(1)设计引物，如下
(2)PCR反应，20μL PCR反应体系如下
PCR的反应条件是：95℃预变性5min，95℃变性30s，58℃退火30s，72℃延伸30s，共进行32次循环，最后72℃再延伸5min。
(3)PCR产物测序验证：PCR产物切胶纯化回收后上样至ABI 3730测序仪进行测序，对结果进行比对分析，具体步骤如下：
①观察测序图谱，峰形标准，波峰与波谷清晰，峰与峰之间距离均匀，底部没有杂峰干扰作为测序结果准确标志。
②参照NCBI数据库中基因的核苷酸序列，将测序结果与目的基因的核苷酸片段序列进行比对，确定碱基序列测序准确性并确定碱基改变位置。
③将家系成员中患病者和正常人的DNA样品测序结果仔细汇总，判断是否符合基因型-表型共分离现象。
结果分析：全外显子组测序筛选出4个候选致病基因(PIKfyve、NPHS1、FPR1、EPG5)，并对所有家系成员Sanger法测序验证及家系共分离分析，最终明确了该家系的致病基因为PIKfyve。结果如图3所示，其突变位于第2条染色体上的第39号外显子，第5828号碱基上的鸟嘌呤突变为腺嘌呤(c.G5828A)，导致对应编码序列上的甘氨酸突变为谷氨酸(p.G1943E)。该突变是一个新致病突变。
实施例2
利用CRISP/Cas9技术构建突变斑马鱼动物模型
构建CRISPR/Cas9载体，包括PIKfyve基因靶向gRNA(guide RNA)等，共同经显微注射于斑马鱼受精卵，通过常规测序、Westernblot与PCR验证幼年期斑马鱼突变形式，明确DNA突变类型，确定模型构建成功，获得PIKfyve突变的斑马鱼模型，如图4所示。
对本领域的技术人员来说，可如以上描述的技术方案以及构思，做出其它各种相应的改变以及形变，而所有的这些改变以及形变都应该属于本发明权利要求的保护范围之内。
序列表
<110> 赵军，深圳市眼科医院
<120> 一种突变PIKfyve基因及其应用
<130> 赵军，深圳市眼科医院
<141> 2020-10-27
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Claims (2)
1.一种先天性白内障的致病基因在制备CC动物模型、诊断试剂、检测/诊断试剂盒中的应用，所述先天性白内障的致病基因与人类野生型PIKfyve基因存在一个突变的差异，突变位于PIKfyve基因的第39号外显子中c.5828G-A，突变的基因序列为SEQ ID NO.1。
2.如权利要求1所述的应用，其特征在于，所述诊断试剂包括PCR反应体系；所述PCR反应体系如下：
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| CN111607641A (zh) * | 2019-02-26 | 2020-09-01 | 中国人民解放军第四军医大学 | 致先天性膜性白内障的新基因突变位点、检测方法及应用 |
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