CN116083458B - 黏多糖贮积症iiic型的致病突变基因及其应用 - Google Patents
黏多糖贮积症iiic型的致病突变基因及其应用 Download PDFInfo
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Abstract
本发明涉及一种黏多糖贮积症IIIC型(MPSIIIC)致病基因新突变及其应用,首次发现一种MPSIIIC致病基因HGSNAT的新突变位点,其中:在先证者HGSNAT基因上检测到复合杂合突变:新发现变异位于7号外显子的错义突变,该突变造成HGSNAT蛋白248位的甘氨酸Gly突变为谷氨酸Glu。本发明进一步分别通过生物信息学和实验手段,验证了两个变异导致HGSNAT的活性缺失以及溶酶体定位失败,由此证实两个变异均为有害致病变异。因此,本发明新致病突变扩展了MPSIIIC致病基因突变谱,为阐明MPSIIIC的发病机制以及开展基因诊断具有重要意义。
Description
技术领域
本发明涉及罕见遗传病MPSIIIC的致病基因HGSNAT突变位点及其在诊断中的应用,属于生物医学领域。
背景技术
黏多糖贮积症(Mucopolysaccharidosis,MPS)是由于溶酶体水解酶的缺乏使不同的酸性黏多糖(氨基葡聚糖)不能完全降解,在各种组织内沉积而引起的临床症状不完全相同的一组疾病。黏多糖的降解必须在溶酶体中进行,已知有10种酶参与其降解过程,其中任何一种酶的缺陷都会造成氨基葡聚糖链的分解障碍而积聚体内并自尿中排出,引起细胞结构异常和功能异常,造成器官的病理改变和临床症状。根据临床表现和酶缺陷,MPS可以分为I~VII等7大类型,每型又细分为若干亚型。
其中,黏多糖贮积症IIIC(Sanfilippo C,MPSIIIC,OMIM#252930)是一种以进行性中枢神经***退化为特征的多***MPS,表现为常染色体隐性遗传,为HGSNAT单基因遗传病,临床症状主要为严重智力障碍、发育退化和其他神经***表现(包括自闭症谱系障碍、行为问题和睡眠障碍)。该病通常在十岁之前发病,患者其他临床表现还包括肌肉/骨骼问题(包括关节僵硬、挛缩、脊柱侧弯和髋关节发育不良等)、听力损失、呼吸道和窦肺感染以及心脏问题(瓣膜增厚、心脏传导***缺陷)等。该病临床异质性高,表型严重程度甚至在同一家族成员之间也不同。
MPSIII型的各种酶缺乏(可有4种不同的酶缺乏)均可引起硫酸类肝素(Heparansulfate,HS)的降解障碍,其中MPSIIIC主要是由乙酰辅酶A:α-氨基葡糖苷N-乙酰转移酶(HGSNAT)基因的致病性变异导致酶活丧失造成的。CN110184337A公开了检测55种遗传病相关的基因和基因位置,涉及黏多糖贮积症的具体为:
疾病亚型 | 基因 | 基因位置 |
黏多糖贮积症1s/Ih/s型 | IDUA | 4P16.3 |
黏多糖贮积症IIIA型 | SGSH | 17q25.3 |
黏多糖贮积症IIIB型 | NAGLU | 17q21.2 |
黏多糖贮积症IIID型 | GNS | 12q14.3 |
黏多糖贮积症II型 | IDS | Xq28 |
此外,还公开了相应的探针和检测试剂盒,未涉及MPSIIIC型检测。
CN112813156A公开了检测诊断骨骼发育障碍致病基因的DNA文库及其应用,该DNA文库包括507个致病基因,但未公开与MPSIIIC相关特异性致病基因。
CN110423805A公开了用于检测新生儿黏多糖贮积症基因分型的引物池,所述引物池包括用于分别特异性扩增GLB1、HYAL1、IDUA、ARSB、GUSB、HGSNAT、IDS、GNS、GALNS、NAGLU、SGSH基因的靶向序列的若干个引物对。其中以HGSNAT为MPSIIIC相关检测基因,但未涉及该基因中的致病突变研究。
综上,目前MPSIIIC的HGSNAT基因突变/变异谱尚未完全发现,基因型和表型的关系尚不完全明确;因此,关于MPSIIIC的基因研究工作仍需不断深入。
发明内容
针对现有技术存在的问题,本发明的目的是确定MPSIIIC致病基因HGSNAT的相关新致病突变点,并分别通过生物信息学和实验手段,验证该突变导致HGSNAT的酶活缺陷以及对溶酶体功能的影响,由此确证该变异位点为有害致病变异,为MPSIIIC的诊断和治疗相关研究提供指导。
基于上述原因,本发明提供一种核苷酸,该核苷酸与MPSIIIC相关,该核苷酸与野生型HGSNAT基因相比,存在c.743G>A;换言之,或该核苷酸与野生型HGSNAT基因相比,该突变的HGSNAT(NM_152419.3)第7外显子含有一个错义突变位点c.743G>A。
优选地,上述核苷酸序列与野生型HGSNAT核苷酸序列相比748位不同,更具体为第743位核苷酸由G突变为A。
优选地,上述核苷酸序列如SEQ ID NO:7所示。
优选地,野生型HGSNAT核苷酸序列如SEQ ID NO:5所示。
作为一种优选方式,上述突变的HGSNAT核酸序列或本发明第二方面所述的蛋白氨基酸序列来源于人或非人哺乳动物,较佳地来源于人。
为实现上述发明目的之二,本发明提供一种蛋白,该蛋白与MPSIIIC相关,该蛋白与野生型HGSNAT蛋白相比,第248位氨基酸由甘氨酸(Gly)为谷氨酸(Glu)。
优选地,上述突变蛋白氨基酸序列如SEQ ID NO:11所示。
作为一种优选方案,本发明第一方面所述的突变的HGSNAT核苷酸序列或本发明第二方面所述的蛋白氨基酸序列来源于人或非人哺乳动物,较佳地来源于人。
为实现上述发明目的之三,本发明还提供了任一上述的核苷酸或蛋白用于制备MPSIIIC检测试剂或检测设备中的应用。
优选地,上述检测试剂选自:引物或引物对、探针、抗体、或核酸芯片中的一种或多种。
更优选地,上述引物或引物对特异性扩增出含743位G的扩增产物;较佳地,扩增产物的长度为100-1000bp。
更优选地,上述的探针可以特异性结合于含743位G的核酸片段。
更优选地,上述的抗体可以特异性结合于含248位突变谷氨酸的多肽。
更优选地,上述试剂为PCR检测743核苷酸位点的试剂。
为实现上述发明目的之四,本发明还提供了检测MPSIIIC的试剂,该试剂至少包括检测检测HGSNAT基因第743位核苷酸位点的试剂,该突变的HGSNAT基因(NM_152419.3)第7外显子含有一个错义突变位点c.743G>A,上述突变的HGSNAT基因第743位核苷酸由G突变为A,其他部分与野生型相同;或检测HGSNAT蛋白第248位氨基酸位点的试剂。
优选地,所述检测HGSNAT基因第743位核苷酸位点的试剂,所述试剂选自下组:引物对、探针、核酸芯片。
优选地,检测HGSNAT蛋白第248位氨基酸位点的试剂为抗体。
优选地,所述的引物对序列中,上游引物如SEQ ID NO:1所示,下游引物如SEQ IDNO:2的引物,更优选地,还包括第二引物对,上游引物如SEQ ID NO:3所示,下游引物如SEQID NO:4所示。
作为本发明目的之五,本发明提供了MPSIIIC型的致病突变基因,该致病突变基因为突变的与野生型HGSNAT基因,该致病突变基因的第7外显子含有一个错义突变位点c.743G>A。
优选地,该致病突变的第743位核苷酸由G突变为A。
优选地,该致病突变的核苷酸序列如SEQ ID NO:7所示。
优选地,该致病突变基因的蛋白第248位氨基酸由甘氨酸(Gly)为谷氨酸(Glu)。
优选地,该致病突变基因的蛋白氨基酸序列如SEQ ID NO:11所示。
术语
外显子:如本文所用,“外显子”一词是指在成熟信使RNA(mRNA)中被保留下的部分,即成熟mRNA对应于基因中的部分。内含子是在mRNA加工过程中被剪切掉的部分,在成熟mRNA中不存在。外显子和内含子都是对于基因而言的,编码的部分为外显子,不编码的为内含子。
引物:如本文所用,术语“引物”指的是能与模板互补配对,在DNA聚合酶的作用合成与模板互补的DNA链的寡聚核苷酸的总称。引物可以是天然的RNA、DNA,也可以是任何形式的天然核苷酸,引物甚至可以是非天然的核苷酸如LNA或ZNA等。引物“大致上”(或“基本上”)与模板上一条链上的一个特殊的序列互补。引物必须与模板上的一条链充分互补才能开始延伸,但引物的序列不必与模板的序列完全互补。比如,在一个3’端与模板互补的引物的5’端加上一段与模板不互补的序列,这样的引物仍大致上与模板互补。只要有足够长的引物能与模板充分的结合,非完全互补的引物也可以与模板形成引物-模板复合物,从而进行扩增。
本领域技术人员应当理解的是,在此,本申请已经对突变位点的信息做了具***置及突变类型的阐明,因此,适用于本申请的核酸包括但不限于基因组DNA(gDNA)、mRNA、互补于mRNA的DNA(cDNA),同时,本申请中的核酸序列可以是互补双链中的任一条或两条。基于核酸序列的互补性和本申请提供的核酸序列信息,本领域技术人员可在明确了一条核酸序列的情况下得到与其互补的另一条核酸序列。关于上述分离的核酸,可以是通过提取、纯化的方式从样品获得,也可以是通过人工合成或人工突变等方式得到,可在检测、治疗、药物开发或研究等PID相关领域中自由使用。例如一种直接的应用方式是,通过对该核酸进行检测,得到样品中是否存在本申请提供的致病突变的信息。需要补充说明的是,野生型HGSNAT基因的gDNA和cDNA序列的具体信息可通过在NCBI网站上分别搜索Gene ID:138050和CCDS47852.1获得。
要说明的是,关于上述致病突变在待测样品中的检测方法,一般包括如下步骤:
样品处理、检测、结果判定。其中,样品处理,是指对待测样品进行相关处理,处理的具体方式根据实际检测的对象也可称为检测目标而定,例如若对样品中的核酸进行检测,此时检测目标为核酸,则样品的处理方式为从样品中将核酸提取出来。而样品本身的类型同样根据检测目标的类型进行选择,例如检测目标为核酸时,样品包括但不限于血液、上皮组织等,通常情况下,相关样本中包括检测目标即可。检测这一步骤也是根据所选的检测目标而定的,检测目标不同时,检测的具体方式也有所区别。例如当检测目标为核酸时,可采用测序的方式来确定样品核酸的序列信息,而测序的具体方法并不构成对本申请的限制,本领域技术人员可基于本申请的提示选择合适的测序方式。在本申请的一种具体实施方式中,采用Sanger测序的方式对样品中的核酸进行检测。将从样品中提取的核酸通过引物进行PCR扩增,对扩增后的片段进行Sanger测序。结果判定,是指将检测的结果与一定的标准进行比对,得出结论。结果判定的方式同样与检测目标的选择相关。在本申请的一个具体实施方式中,采用将测序结果与标准序列进行比对,若待测样品的核酸序列中含有本申请公开的两种致病突变中的一种,即为阳性结果,否则为阴性。
与现有技术相比,本发明首次发现了单基因遗传病MPSIIIC的致病基因HGSNAT中的新致病突变,该致病突变也是首次在中国人群发现,通过本发明提供的检测试剂也在患病家族系中得到验证,此外,本发明还通过实验验证了该突变致病性,并探讨了相应致病机理。该致病突变,不仅扩展了MPSIIIC致病基因突变谱,同时为开展基因诊断提供了新数据,为该疾病的诊断提供了新的分子生物学基础。该新致病突变位点的发现可以对其家系后代进行优生优育指导,并可作为人群范围内基因诊断携带者筛查和产前诊断筛查位点,从而帮助了解发病机制、辅助临床诊断、产前诊断和转基因治疗,为我国黏多糖贮积症防治提供了新的研究方向。
附图说明
图1为先证者的影像图片;
图2为实施例中MPSIIIC家系图;
图3为实施例中先证者及父母基因型Sanger测序结果;
图4为实施例中致病突变的保守性分析结果图;
图5为PUC57-HGSNAT::MYC::FLAG克隆载体;
图6为pCSC-IRES-GFP表达载体;
图7为pCSC-HGSNAT-WT::MYC::FLAG表达载体;
图8为pCSC-HGSNAT-R344C::MYC::FLAG表达载体;
图9为pCSC-HGSNAT-G248E::MYC::FLAG表达载体;
图10为pCSC-HGSNAT-P237Q::MYC::FLAG表达载体;
图11为293T细胞中分别过表达野生型(WT)、R344C、G248E、P237Q的全长HGSNAT::MYC::FLAG质粒,裂解细胞以后利用SDS-PAGE检测蛋白表达结果图;
图12为293T细胞中分别过表达野生型(WT)、R344C、G248E的全长HGSNAT::MYC::FLAG质粒,裂解细胞以后分别测定HGSNAT酶活性以及对照NAGase酶活性图;
图13为pLAMP1-mCherry报告载体;
图14为293T细胞中分别过表达野生型(WT)、R344C、G248E、P237Q的全长HGSNAT::MYC::FLAG质粒,以及溶酶体定位的报告质粒pLAMP1-mCherry,使用免疫荧光检测HGSNAT在溶酶体中的定位结果图。
具体实施方式
下面结合实施例对本发明提供的单克隆抗体的制备方法及其应用作进一步详细、完整地说明。下面描述的实施例是示例性的,仅用于解释本发明,而不能理解为对本发明的限制。
下述实施例中的实验方法,如无特殊说明,通常按照常规条件如J.萨姆布鲁克等编著,分子克隆实验指南,第三版,科学出版社,2002中所述的条件,或按照制造厂商所建议的条件。如无特殊说明,本发明实施例中所涉及的试剂均为市售产品,均可以通过商业渠道购买获得。
本发明实施例涉及的主要生物材料如下:
DMEM完全培养基:DMEM,10%胎牛血清(FBS)和1%青链霉素混合液
青链霉素混合液(Solarbio,Beijing,China,#P1400)
4% PFA(Servicebio,Wuhan,China,#G1101)
Triton X-100(Solarbio,#T8200)
Tween-20(Macklin,Rochelle,IL,USA,#C10232628)
Bovine serum albumin(BSA,BioFroxx,Guangzhou,China,#4240)
Hoechst 33342(Beyotime,Shanghai,China,#C1025)
Poly(vinyl alcohol)(PVA,Macklin,#P816862)
封闭液:3% BSA,2% Triton X-100,DPBS
TBS(10x):Tris-base 48.4g及NaCl 160g溶至0.8L水,用浓盐酸调pH至7.6
LB液体培养基:蛋白胨10g,酵母提取物5g,NaCl 10g(定容至1L,高压蒸汽灭菌,4℃保存)
TAE缓冲液(10x):Tris-base 24.2g,EDTA 5.71g,冰乙酸5.71ml(定容至1L)
McIlvain缓冲液(pH 5.5,源叶生物,#R20258)
甘氨酸缓冲液(0.4M):1.5g甘氨酸溶于50ml水,用1N NaOH调pH至10.4
4-甲基伞酮-β-D半乳糖苷(MU-βGlcNH2,Biosynth,#EM31025)
4-甲基蛋白酚对乙酰-β-D氨基葡萄糖(MU-βGlcNAc,Biosynth,#M5504)
4-甲基伞酮(4-MU,Sigma,#M1381)
乙酰辅酶A(Sigma,#A2056)
分子克隆所用酶和试剂型号和来源如表1所示
表1.分子克隆用酶及试剂
实验材料 | 货号 | 来源 |
Gel-Green核酸染料 | #SCT125 | Merck |
AgeI-HF | #R3552 | NEB |
BsrG1-HF | #R3575 | NEB |
DpnI | #R0176 | NEB |
PrimeSTAR HS DNA聚合酶 | #R010A | TAKARA |
T4连接酶 | #EL0011 | Thermo |
免疫荧光染色和SDS-PAGE所用一抗和二抗的型号和来源如表2所示。
表2.免疫荧光染色和SDS-PAGE所用一抗和二抗
实施例致病基因突变位点的鉴定及验证
一、致病基因突变位点的筛查
1、研究人群
研究队列:选取一个3代MPSIIIC家系,先证者II6,女性,15岁,临床上开展双髋部疼痛查因,智力低下查因。先证者5年前出现双髋部疼痛不适,严重时无法行走。X射线检查显示其双侧股骨头变扁,双侧髋关节面下见多发囊性低密度改变,以及双侧骶骼关节面骨质密度增高,同时脊柱生理曲度存在,椎体序列稍不连续,T10、T11椎体下缘见许莫氏结节形成,相应椎体轻度变扁(影像图片见图1,其中A:先证者的X射线检查显示其双侧股骨头变扁,双侧髋关节面下见多发囊性低密度改变,以及双侧骶骼关节面骨质密度增高;B:先证者的X射线检查显示其脊柱生理曲度存在,椎体序列稍不连续,T10、T11椎体下缘见许莫氏结节形成,相应椎体轻度变扁)。此外,先证者目前上小学6年级,成绩较差,平日无法自读,提示其存在严重智力缺陷。其父母表型均正常,非近亲结婚;先证者有一姐姐,表型正常,有两子女均正常,先证者母亲自诉另有三子女均亡故。家系图见图2,其中黑色箭头示先证者;黑色符号表示受累状态;斜线表示已过世。按照按照知情同意的原则,在先证者及其家属自愿签署知情同意书的前提下,寄送5-10mL外周血样本,建立病历资料库,详细记录先证者病情、家系情况等资料。本研究已得到本单位伦理委员会批准。
2、研究方法
2.1对先证者进行外显子组测序
从受检者样本中提取基因组gDNA,构建基因组文库。通过探针杂交捕获目标基因(约20,000个)外显子及毗邻剪接区域(约20bp),并进行富集。对富集的基因进行质量控制,利用Illumina高通量测序仪进行测序。本次受检样本目标区域捕获测序参数如表3所示:
表3样本目标区域捕获测序参数
样本编号 | 原始数据量(G) | 平均测序深度 | ≥10X覆盖度 | Q30(%) |
07W210909GDT6075801B01 | 13.92 | 182.14 | 99.71 | 91.87 |
2.2分析
测序原始数据首先去除不符合质控要求的reads,然后运用BWA软件与UCSC提供的hg19版本人类基因组参考序列进行比对,经过GATK的HaplotypeCaller找出其中的SNV和InDel变异,再经过下列专业数据库和生信预测软件,以及韦翰斯自有的本地数据库和分析软件进行进一步注释和筛选。采用xhmm和clamms算法对探针覆盖区域进行拷贝数变异分析。
人群变异频率数据库:1000Genomes、ESP、ExAC、gnomAD等;
位点及疾病数据库:dbSNP、OMIM、HGMD、ClinVar、Decipher、DGV等;
生信预测软件:SIFT、Polyphen2、LRT、MutationTaster、FATHMM、M-CAP、CADD、REVEL、dbscSNV等。
2.3解读:
序列变异数据解读规则参考美国医学遗传学和基因组学学会(American Collegeof Medical Genetics and Genomics,ACMG)遗传变异分类标准与指南,以及ClinGen序列变异解读工作组,耳聋、心肌病、苯丙酮尿症等专家组等发布的序列变异解读指南(指南来源ClinGen官网)进行进一步的细化解读。拷贝数变异(CNV)解读规则参考2019版ACMG拷贝数变异解读和报告指南。
2.4变异解释
在先证者HGSNAT基因上检测到2个杂合变异,错义变异1:c.1030C>T(p.Arg344Cys),错义变异2:c.743G>A(p.Gly248Glu)。变异1导致第344位氨基酸由精氨酸变异为半胱氨酸,变异2导致第248位氨基酸由甘氨酸变异为谷氨酸(详见图3,显示变异1:c.1030C>T(p.Arg344Cys)源自父亲,变异2:c.743G>A(p.Gly248Glu)源自母亲,先证者为复合杂合突变)。通过对先证者二代测序数据进行生物信息学分析、拷贝数变异分析及结合临床表型的综合分析,未发现与先证者检测目的相关的已知/可能具有临床意义的CNVs。
有多篇文献报道在多例MPSIIIC患者中检测到变异1,该变异收录于HGMD数据库(CM065262),ClinVar数据库有1条记录显示该变异为可能致病性变异,有4条记录显示该变异为致病性变异。变异2暂无文献报道,参照ACMG相关指南,变异1为致病变异,变异2为临床意义未明变异。使用生信预测软件如SIFT、Polyphen2、MutationTaster、CADD分析变异致病性,均显示变异2可能是致病性变异;另外,HGSNAT p.P237Q变异为已报道的基因多态性,不影响HGSNAT酶活性,用于对照。In silico分析结果见表4:
表4.In silico预测HGSNAT突变的致病性
此外,对变异位点开展保守性分析,具体地,对脊椎动物氨基酸序列使用WebLogo软件进行分析,分析结果显示R344以及G248位点均高度保守(详见图4),说明两个位点在HGSNAT蛋白组成和功能发挥上的重要性。
二、致病基因突变位点的鉴定
采用本发明提供的检测试剂盒对同一先证者家族成员进行致病突变的鉴定。
本实施例还公开了一种用于检测致病突变的试剂,该试剂包括以下两组引物对,用于扩增。
第一组引物对,可检测HGSNAT基因上的突变p.G248E:
上游引物:Ex7-F:CTGAAGGAGCTGGGATCTCCC(SEQ ID NO:1)
下游引物:Ex7-R:AGTGGACACTGGCTCTGGCCT(SEQ ID NO:2)
第二组引物对,可检测HGSNAT基因上的突变p.R344C:
上游引物:Ex11-F:GCCATGTCCCTGACTGACCCT(SEQ ID NO:3)
下游引物:EX11-R:CTGGGCAACACAGCAAGACCC(SEQ ID NO:4)
还公开了该检测试剂的具体检测步骤:
1)样品处理:采集先证者及其家系(共四人)的外周血,经红细胞裂解后提取gDNA。
2)检测:使用PCR分别扩增2个突变位点周边区域,经跑TAE核酸胶以后,切胶送Sanger测序;其中扩增相关步骤如下:150ng待测gDNA加入到50μL反应体系中,反应体系由5x PrimeSTAR Buffer(Mg2+plus)10μL,dNTP(2.5μM)4μL,正向引物和反向引物(10μM)各1.25μL,PrimeSTAR HS DNA聚合酶(2.5units/μL)0.5μL,Betaine(4M)12.5μL,补水使总体积为50μL。PCR反应程序为:预变性阶段94℃,60秒;变性阶段98℃,10秒;退火阶段56℃,15秒;延伸阶段72℃,1min/1kb;冷却4℃,10min;变性,退火,延伸三个阶段循环35次,即完成扩增。
得到的PCR产物跑TAE胶,后对照PCR产物大小,切胶送Sanger测序(北京擎科测序公司)。
测序后HGSNAT核苷酸序列如下:
野生型HGSNAT CDS核苷酸序列:
ATGAGCGGGGCGGGCAGGGCGCTGGCCGCGCTGCTGCTGGCCGCGTCCGTGCTGAGCGCCGCGCTGCTGGCCCCCGGCGGCTCTTCGGGGCGCGATGCCCAGGCCGCGCCGCCACGAGACTTAGACAAAAAAAGACATGCAGAGCTGAAGATGGATCAGGCTTTGCTACTCATCCATAATGAACTTCTCTGGACCAACTTGACCGTCTACTGGAAATCTGAATGCTGTTATCACTGCTTGTTTCAGGTTCTGGTAAACGTTCCTCAGAGTCCAAAAGCAGGGAAGCCTAGTGCTGCAGCTGCCTCTGTCAGCACCCAGCACGGATCTATCCTGCAGCTGAACGACACCTTGGAAGAGAAAGAAGTTTGTAGGTTGGAATACAGATTTGGAGAATTTGGAAACTATTCTCTCTTGGTAAAGAACATCCATAATGGAGTTAGTGAAATTGCCTGTGACCTGGCTGTGAACGAGGATCCAGTTGATAGTAACCTTCCTGTGAGCATTGCATTCCTTATTGGTCTTGCTGTCATCATTGTGATATCCTTTCTGAGGCTCTTGTTGAGTTTGGATGACTTTAACAATTGGATTTCTAAAGCCATAAGTTCTCGAGAAACTGATCGCCTCATCAATTCTGAGCTGGGATCTCCCAGCAGGACAGACCCTCTCGATGGTGATGTTCAGCCAGCAACGTGGCGTCTATCTGCCCTGCCGCCCCGCCTCCGCAGCGTGGACACCTTCAGGGGGATTGCTCTTATACTCATGGTCTTTGTCAATTATGGAGGAGGAAAATATTGGTACTTCAAACATGCAAGTTGGAATGGGCTGACAGTGGCTGACCTCGTGTTCCCGTGGTTTGTATTTATTATGGGATCTTCCATTTTTCTATCGATGACTTCTATACTGCAACGGGGGTGTTCAAAATTCAGATTGCTGGGGAAGATTGCATGGAGGAGTTTCCTGTTAATCTGCATAGGAATTATCATTGTGAATCCCAATTATTGCCTTGGTCCATTGTCTTGGGACAAGGTGCGCATTCCTGGTGTGCTGCAGCGATTGGGAGTGACATACTTTGTGGTTGCTGTGTTGGAGCTCCTCTTTGCTAAACCTGTGCCTGAACATTGTGCCTCGGAGAGGAGCTGCCTTTCTCTTCGAGACATCACGTCCAGCTGGCCCCAGTGGCTGCTCATCCTGGTGCTGGAAGGCCTGTGGCTGGGCTTGACATTCCTCCTGCCAGTCCCTGGGTGCCCTACTGGTTATCTTGGTCCTGGGGGCATTGGAGATTTTGGCAAGTATCCAAATTGCACTGGAGGAGCTGCAGGCTACATCGACCGCCTGCTGCTGGGAGACGATCACCTTTACCAGCACCCATCTTCTGCTGTACTTTACCACACCGAGGTGGCCTATGACCCCGAGGGCATCCTGGGCACCATCAACTCCATCGTGATGGCCTTTTTAGGAGTTCAGGCAGGAAAAATACTATTGTATTACAAGGCTCGGACCAAAGACATCCTGATTCGATTCACTGCTTGGTGTTGTATTCTTGGGCTCATTTCTGTTGCTCTGACGAAGGTTTCTGAAAATGAAGGCTTTATTCCAGTAAACAAAAATCTCTGGTCCCTTTCGTATGTCACTACGCTCAGTTCTTTTGCCTTCTTCATCCTGCTGGTCCTGTACCCAGTTGTGGATGTGAAGGGGCTGTGGACAGGAACCCCATTCTTTTATCCAGGAATGAATTCCATTCTGGTATATGTCGGCCACGAGGTGTTTGAGAACTACTTCCCCTTTCAGTGGAAGCTGAAGGACAACCAGTCCCACAAGGAGCACCTGACTCAGAACATCGTCGCCACTGCCCTCTGGGTGCTCATTGCCTACATCCTCTATAGAAAGAAGATTTTTTGGAAAATCTGA(SEQ ID NO:5)
突变1HGSNAT-R344C CDS核苷酸序列:
ATGAGCGGGGCGGGCAGGGCGCTGGCCGCGCTGCTGCTGGCCGCGTCCGTGCTGAGCGCCGCGCTGCTGGCCCCCGGCGGCTCTTCGGGGCGCGATGCCCAGGCCGCGCCGCCACGAGACTTAGACAAAAAAAGACATGCAGAGCTGAAGATGGATCAGGCTTTGCTACTCATCCATAATGAACTTCTCTGGACCAACTTGACCGTCTACTGGAAATCTGAATGCTGTTATCACTGCTTGTTTCAGGTTCTGGTAAACGTTCCTCAGAGTCCAAAAGCAGGGAAGCCTAGTGCTGCAGCTGCCTCTGTCAGCACCCAGCACGGATCTATCCTGCAGCTGAACGACACCTTGGAAGAGAAAGAAGTTTGTAGGTTGGAATACAGATTTGGAGAATTTGGAAACTATTCTCTCTTGGTAAAGAACATCCATAATGGAGTTAGTGAAATTGCCTGTGACCTGGCTGTGAACGAGGATCCAGTTGATAGTAACCTTCCTGTGAGCATTGCATTCCTTATTGGTCTTGCTGTCATCATTGTGATATCCTTTCTGAGGCTCTTGTTGAGTTTGGATGACTTTAACAATTGGATTTCTAAAGCCATAAGTTCTCGAGAAACTGATCGCCTCATCAATTCTGAGCTGGGATCTCCCAGCAGGACAGACCCTCTCGATGGTGATGTTCAGCCAGCAACGTGGCGTCTATCTGCCCTGCCGCCCCGCCTCCGCAGCGTGGACACCTTCAGGGGGATTGCTCTTATACTCATGGTCTTTGTCAATTATGGAGGAGGAAAATATTGGTACTTCAAACATGCAAGTTGGAATGGGCTGACAGTGGCTGACCTCGTGTTCCCGTGGTTTGTATTTATTATGGGATCTTCCATTTTTCTATCGATGACTTCTATACTGCAACGGGGGTGTTCAAAATTCAGATTGCTGGGGAAGATTGCATGGAGGAGTTTCCTGTTAATCTGCATAGGAATTATCATTGTGAATCCCAATTATTGCCTTGGTCCATTGTCTTGGGACAAGGTGTGCATTCCTGGTGTGCTGCAGCGATTGGGAGTGACATACTTTGTGGTTGCTGTGTTGGAGCTCCTCTTTGCTAAACCTGTGCCTGAACATTGTGCCTCGGAGAGGAGCTGCCTTTCTCTTCGAGACATCACGTCCAGCTGGCCCCAGTGGCTGCTCATCCTGGTGCTGGAAGGCCTGTGGCTGGGCTTGACATTCCTCCTGCCAGTCCCTGGGTGCCCTACTGGTTATCTTGGTCCTGGGGGCATTGGAGATTTTGGCAAGTATCCAAATTGCACTGGAGGAGCTGCAGGCTACATCGACCGCCTGCTGCTGGGAGACGATCACCTTTACCAGCACCCATCTTCTGCTGTACTTTACCACACCGAGGTGGCCTATGACCCCGAGGGCATCCTGGGCACCATCAACTCCATCGTGATGGCCTTTTTAGGAGTTCAGGCAGGAAAAATACTATTGTATTACAAGGCTCGGACCAAAGACATCCTGATTCGATTCACTGCTTGGTGTTGTATTCTTGGGCTCATTTCTGTTGCTCTGACGAAGGTTTCTGAAAATGAAGGCTTTATTCCAGTAAACAAAAATCTCTGGTCCCTTTCGTATGTCACTACGCTCAGTTCTTTTGCCTTCTTCATCCTGCTGGTCCTGTACCCAGTTGTGGATGTGAAGGGGCTGTGGACAGGAACCCCATTCTTTTATCCAGGAATGAATTCCATTCTGGTATATGTCGGCCACGAGGTGTTTGAGAACTACTTCCCCTTTCAGTGGAAGCTGAAGGACAACCAGTCCCACAAGGAGCACCTGACTCAGAACATCGTCGCCACTGCCCTCTGGGTGCTCATTGCCTACATCCTCTATAGAAAGAAGATTTTTTGGAAAATCTGA(SEQ ID NO:6)
突变2HGSNAT-G248E CDS核苷酸序列:
ATGAGCGGGGCGGGCAGGGCGCTGGCCGCGCTGCTGCTGGCCGCGTCCGTGCTGAGCGCCGCGCTGCTGGCCCCCGGCGGCTCTTCGGGGCGCGATGCCCAGGCCGCGCCGCCACGAGACTTAGACAAAAAAAGACATGCAGAGCTGAAGATGGATCAGGCTTTGCTACTCATCCATAATGAACTTCTCTGGACCAACTTGACCGTCTACTGGAAATCTGAATGCTGTTATCACTGCTTGTTTCAGGTTCTGGTAAACGTTCCTCAGAGTCCAAAAGCAGGGAAGCCTAGTGCTGCAGCTGCCTCTGTCAGCACCCAGCACGGATCTATCCTGCAGCTGAACGACACCTTGGAAGAGAAAGAAGTTTGTAGGTTGGAATACAGATTTGGAGAATTTGGAAACTATTCTCTCTTGGTAAAGAACATCCATAATGGAGTTAGTGAAATTGCCTGTGACCTGGCTGTGAACGAGGATCCAGTTGATAGTAACCTTCCTGTGAGCATTGCATTCCTTATTGGTCTTGCTGTCATCATTGTGATATCCTTTCTGAGGCTCTTGTTGAGTTTGGATGACTTTAACAATTGGATTTCTAAAGCCATAAGTTCTCGAGAAACTGATCGCCTCATCAATTCTGAGCTGGGATCTCCCAGCAGGACAGACCCTCTCGATGGTGATGTTCAGCCAGCAACGTGGCGTCTATCTGCCCTGCCGCCCCGCCTCCGCAGCGTGGACACCTTCAGGGAGATTGCTCTTATACTCATGGTCTTTGTCAATTATGGAGGAGGAAAATATTGGTACTTCAAACATGCAAGTTGGAATGGGCTGACAGTGGCTGACCTCGTGTTCCCGTGGTTTGTATTTATTATGGGATCTTCCATTTTTCTATCGATGACTTCTATACTGCAACGGGGGTGTTCAAAATTCAGATTGCTGGGGAAGATTGCATGGAGGAGTTTCCTGTTAATCTGCATAGGAATTATCATTGTGAATCCCAATTATTGCCTTGGTCCATTGTCTTGGGACAAGGTGCGCATTCCTGGTGTGCTGCAGCGATTGGGAGTGACATACTTTGTGGTTGCTGTGTTGGAGCTCCTCTTTGCTAAACCTGTGCCTGAACATTGTGCCTCGGAGAGGAGCTGCCTTTCTCTTCGAGACATCACGTCCAGCTGGCCCCAGTGGCTGCTCATCCTGGTGCTGGAAGGCCTGTGGCTGGGCTTGACATTCCTCCTGCCAGTCCCTGGGTGCCCTACTGGTTATCTTGGTCCTGGGGGCATTGGAGATTTTGGCAAGTATCCAAATTGCACTGGAGGAGCTGCAGGCTACATCGACCGCCTGCTGCTGGGAGACGATCACCTTTACCAGCACCCATCTTCTGCTGTACTTTACCACACCGAGGTGGCCTATGACCCCGAGGGCATCCTGGGCACCATCAACTCCATCGTGATGGCCTTTTTAGGAGTTCAGGCAGGAAAAATACTATTGTATTACAAGGCTCGGACCAAAGACATCCTGATTCGATTCACTGCTTGGTGTTGTATTCTTGGGCTCATTTCTGTTGCTCTGACGAAGGTTTCTGAAAATGAAGGCTTTATTCCAGTAAACAAAAATCTCTGGTCCCTTTCGTATGTCACTACGCTCAGTTCTTTTGCCTTCTTCATCCTGCTGGTCCTGTACCCAGTTGTGGATGTGAAGGGGCTGTGGACAGGAACCCCATTCTTTTATCCAGGAATGAATTCCATTCTGGTATATGTCGGCCACGAGGTGTTTGAGAACTACTTCCCCTTTCAGTGGAAGCTGAAGGACAACCAGTCCCACAAGGAGCACCTGACTCAGAACATCGTCGCCACTGCCCTCTGGGTGCTCATTGCCTACATCCTCTATAGAAAGAAGATTTTTTGGAAAATCTGA(SEQ ID NO:7)
此外,为验证上述两个突变的致病性,采用已报道的多态性突变点为对照,该基因多态性突变HGSNAT-P237Q CDS核苷酸序列:
ATGAGCGGGGCGGGCAGGGCGCTGGCCGCGCTGCTGCTGGCCGCGTCCGTGCTGAGCGCCGCGCTGCTGGCCCCCGGCGGCTCTTCGGGGCGCGATGCCCAGGCCGCGCCGCCACGAGACTTAGACAAAAAAAGACATGCAGAGCTGAAGATGGATCAGGCTTTGCTACTCATCCATAATGAACTTCTCTGGACCAACTTGACCGTCTACTGGAAATCTGAATGCTGTTATCACTGCTTGTTTCAGGTTCTGGTAAACGTTCCTCAGAGTCCAAAAGCAGGGAAGCCTAGTGCTGCAGCTGCCTCTGTCAGCACCCAGCACGGATCTATCCTGCAGCTGAACGACACCTTGGAAGAGAAAGAAGTTTGTAGGTTGGAATACAGATTTGGAGAATTTGGAAACTATTCTCTCTTGGTAAAGAACATCCATAATGGAGTTAGTGAAATTGCCTGTGACCTGGCTGTGAACGAGGATCCAGTTGATAGTAACCTTCCTGTGAGCATTGCATTCCTTATTGGTCTTGCTGTCATCATTGTGATATCCTTTCTGAGGCTCTTGTTGAGTTTGGATGACTTTAACAATTGGATTTCTAAAGCCATAAGTTCTCGAGAAACTGATCGCCTCATCAATTCTGAGCTGGGATCTCCCAGCAGGACAGACCCTCTCGATGGTGATGTTCAGCCAGCAACGTGGCGTCTATCTGCCCTGCAGCCCCGCCTCCGCAGCGTGGACACCTTCAGGGGGATTGCTCTTATACTCATGGTCTTTGTCAATTATGGAGGAGGAAAATATTGGTACTTCAAACATGCAAGTTGGAATGGGCTGACAGTGGCTGACCTCGTGTTCCCGTGGTTTGTATTTATTATGGGATCTTCCATTTTTCTATCGATGACTTCTATACTGCAACGGGGGTGTTCAAAATTCAGATTGCTGGGGAAGATTGCATGGAGGAGTTTCCTGTTAATCTGCATAGGAATTATCATTGTGAATCCCAATTATTGCCTTGGTCCATTGTCTTGGGACAAGGTGCGCATTCCTGGTGTGCTGCAGCGATTGGGAGTGACATACTTTGTGGTTGCTGTGTTGGAGCTCCTCTTTGCTAAACCTGTGCCTGAACATTGTGCCTCGGAGAGGAGCTGCCTTTCTCTTCGAGACATCACGTCCAGCTGGCCCCAGTGGCTGCTCATCCTGGTGCTGGAAGGCCTGTGGCTGGGCTTGACATTCCTCCTGCCAGTCCCTGGGTGCCCTACTGGTTATCTTGGTCCTGGGGGCATTGGAGATTTTGGCAAGTATCCAAATTGCACTGGAGGAGCTGCAGGCTACATCGACCGCCTGCTGCTGGGAGACGATCACCTTTACCAGCACCCATCTTCTGCTGTACTTTACCACACCGAGGTGGCCTATGACCCCGAGGGCATCCTGGGCACCATCAACTCCATCGTGATGGCCTTTTTAGGAGTTCAGGCAGGAAAAATACTATTGTATTACAAGGCTCGGACCAAAGACATCCTGATTCGATTCACTGCTTGGTGTTGTATTCTTGGGCTCATTTCTGTTGCTCTGACGAAGGTTTCTGAAAATGAAGGCTTTATTCCAGTAAACAAAAATCTCTGGTCCCTTTCGTATGTCACTACGCTCAGTTCTTTTGCCTTCTTCATCCTGCTGGTCCTGTACCCAGTTGTGGATGTGAAGGGGCTGTGGACAGGAACCCCATTCTTTTATCCAGGAATGAATTCCATTCTGGTATATGTCGGCCACGAGGTGTTTGAGAACTACTTCCCCTTTCAGTGGAAGCTGAAGGACAACCAGTCCCACAAGGAGCACCTGACTCAGAACATCGTCGCCACTGCCCTCTGGGTGCTCATTGCCTACATCCTCTATAGAAAGAAGATTTTTTGGAAAATCTGA(SEQ ID NO:8)
其中核苷酸测序结果显示,突变1的HGSNAT核苷酸序列的第1030位核苷酸由C突变为T,突变2中的HGSNAT核苷酸序列的第743位核苷酸由G突变为A。
相应的,HGSNAT蛋白氨基酸序列如下:
野生型HGSNAT蛋白氨基酸序列:
MSGAGRALAALLLAASVLSAALLAPGGSSGRDAQAAPPRDLDKKRHAELKMDQALLLIHNELLWTNLTVYWKSECCYHCLFQVLVNVPQSPKAGKPSAAAASVSTQHGSILQLNDTLEEKEVCRLEYRFGEFGNYSLLVKNIHNGVSEIACDLAVNEDPVDSNLPVSIAFLIGLAVIIVISFLRLLLSLDDFNNWISKAISSRETDRLINSELGSPSRTDPLDGDVQPATWRLSALPPRLRSVDTFRGIALILMVFVNYGGGKYWYFKHASWNGLTVADLVFPWFVFIMGSSIFLSMTSILQRGCSKFRLLGKIAWRSFLLICIGIIIVNPNYCLGPLSWDKVRIPGVLQRLGVTYFVVAVLELLFAKPVPEHCASERSCLSLRDITSSWPQWLLILVLEGLWLGLTFLLPVPGCPTGYLGPGGIGDFGKYPNCTGGAAGYIDRLLLGDDHLYQHPSSAVLYHTEVAYDPEGILGTINSIVMAFLGVQAGKILLYYKARTKDILIRFTAWCCILGLISVALTKVSENEGFIPVNKNLWSLSYVTTLSSFAFFILLVLYPVVDVKGLWTGTPFFYPGMNSILVYVGHEVFENYFPFQWKLKDNQSHKEHLTQNIVATALWVLIAYILYRKKIFWKI*(SEQ ID NO:9)
突变1HGSNAT-R344C蛋白氨基酸序列:
MSGAGRALAALLLAASVLSAALLAPGGSSGRDAQAAPPRDLDKKRHAELKMDQALLLIHNELLWTNLTVYWKSECCYHCLFQVLVNVPQSPKAGKPSAAAASVSTQHGSILQLNDTLEEKEVCRLEYRFGEFGNYSLLVKNIHNGVSEIACDLAVNEDPVDSNLPVSIAFLIGLAVIIVISFLRLLLSLDDFNNWISKAISSRETDRLINSELGSPSRTDPLDGDVQPATWRLSALPPRLRSVDTFRGIALILMVFVNYGGGKYWYFKHASWNGLTVADLVFPWFVFIMGSSIFLSMTSILQRGCSKFRLLGKIAWRSFLLICIGIIIVNPNYCLGPLSWDKVCIPGVLQRLGVTYFVVAVLELLFAKPVPEHCASERSCLSLRDITSSWPQWLLILVLEGLWLGLTFLLPVPGCPTGYLGPGGIGDFGKYPNCTGGAAGYIDRLLLGDDHLYQHPSSAVLYHTEVAYDPEGILGTINSIVMAFLGVQAGKILLYYKARTKDILIRFTAWCCILGLISVALTKVSENEGFIPVNKNLWSLSYVTTLSSFAFFILLVLYPVVDVKGLWTGTPFFYPGMNSILVYVGHEVFENYFPFQWKLKDNQSHKEHLTQNIVATALWVLIAYILYRKKIFWKI*(SEQ ID NO:10)
突变2HGSNAT-G248E蛋白氨基酸序列:
MSGAGRALAALLLAASVLSAALLAPGGSSGRDAQAAPPRDLDKKRHAELKMDQALLLIHNELLWTNLTVYWKSECCYHCLFQVLVNVPQSPKAGKPSAAAASVSTQHGSILQLNDTLEEKEVCRLEYRFGEFGNYSLLVKNIHNGVSEIACDLAVNEDPVDSNLPVSIAFLIGLAVIIVISFLRLLLSLDDFNNWISKAISSRETDRLINSELGSPSRTDPLDGDVQPATWRLSALPPRLRSVDTFREIALILMVFVNYGGGKYWYFKHASWNGLTVADLVFPWFVFIMGSSIFLSMTSILQRGCSKFRLLGKIAWRSFLLICIGIIIVNPNYCLGPLSWDKVRIPGVLQRLGVTYFVVAVLELLFAKPVPEHCASERSCLSLRDITSSWPQWLLILVLEGLWLGLTFLLPVPGCPTGYLGPGGIGDFGKYPNCTGGAAGYIDRLLLGDDHLYQHPSSAVLYHTEVAYDPEGILGTINSIVMAFLGVQAGKILLYYKARTKDILIRFTAWCCILGLISVALTKVSENEGFIPVNKNLWSLSYVTTLSSFAFFILLVLYPVVDVKGLWTGTPFFYPGMNSILVYVGHEVFENYFPFQWKLKDNQSHKEHLTQNIVATALWVLIAYILYRKKIFWKI*(SEQ ID NO:11)
基因多态性HGSNAT-P237Q蛋白氨基酸序列:
MSGAGRALAALLLAASVLSAALLAPGGSSGRDAQAAPPRDLDKKRHAELKMDQALLLIHNELLWTNLTVYWKSECCYHCLFQVLVNVPQSPKAGKPSAAAASVSTQHGSILQLNDTLEEKEVCRLEYRFGEFGNYSLLVKNIHNGVSEIACDLAVNEDPVDSNLPVSIAFLIGLAVIIVISFLRLLLSLDDFNNWISKAISSRETDRLINSELGSPSRTDPLDGDVQPATWRLSALQPRLRSVDTFRGIALILMVFVNYGGGKYWYFKHASWNGLTVADLVFPWFVFIMGSSIFLSMTSILQRGCSKFRLLGKIAWRSFLLICIGIIIVNPNYCLGPLSWDKVRIPGVLQRLGVTYFVVAVLELLFAKPVPEHCASERSCLSLRDITSSWPQWLLILVLEGLWLGLTFLLPVPGCPTGYLGPGGIGDFGKYPNCTGGAAGYIDRLLLGDDHLYQHPSSAVLYHTEVAYDPEGILGTINSIVMAFLGVQAGKILLYYKARTKDILIRFTAWCCILGLISVALTKVSENEGFIPVNKNLWSLSYVTTLSSFAFFILLVLYPVVDVKGLWTGTPFFYPGMNSILVYVGHEVFENYFPFQWKLKDNQSHKEHLTQNIVATALWVLIAYILYRKKIFWKI*(SEQ ID NO:12)
其中蛋白氨基酸序列结果显示,突变1的HGSNAT蛋白的第344位精氨酸(Arg)变异为半胱氨酸(Cys),突变2的HGSNAT蛋白的第248位氨基酸由甘氨酸(Gly)突变为谷氨酸(Glu)。
先证者家族测序结果与图3结果一致,其中家族四位成员的受试组基因中两个突变的检测结果如表5所示:
表5突变检测结果
受试者 | 突变1-R344C | 突变2-G248E | 临床症状 |
父亲 | 阳性 | 阴性 | 健康 |
母亲 | 阴性 | 阳性 | 健康 |
姐妹 | 阴性 | 阳性 | 健康 |
先证者 | 阳性 | 阳性 | 致病 |
由表5可知,先证者因同时携带两个致病突变而致病,这符合MPSIIIC是隐性遗传的特征。进一步,这例患者及200名健康对照进行单个突变位点的Sanger测序,发现仅该患者存在该突变位点,而其健康对照均未见该突变位点。值得说明的是,这是发明人首次发现一种致病基因HGSNAT的新突变,这也是首次在中国人群中发现新的致病突变,通过发明人制备的致病突变检测试剂也在该家族系中得到了有力验证。
三、G248突变致病预测
1、HGSNAT的酶活检测
1.1表达载体的构建
构建全长HGSNAT的表达载体。具体地,订购合成的PUC57-HGSNAT::MYC::FLAG克隆载体(如图5所示),以此为模板,用PCR扩增HGSNAT::MYC::FLAG片段,引物对序列为:
上游引物:0583-F:CGCACCGGTAGTGGTACCATGAGCGGG(SEQ ID NO:13)
下游引物:0583-R:CGCTGTACATCACTTGTCGTCATCGTCTTTGT(SEQ ID NO:14)
扩增相关步骤如下:100ng克隆载体加入到50μL反应体系中,反应体系由正向引物和反向引物(10μM)各1.25μL,5 x PrimeSTAR Buffer(Mg2+plus)10μL,dNTP(2.5μM)4μL,PrimeSTAR HS DNA聚合酶(2.5units/μL)0.5μL,补水使总体积为50μL。PCR反应程序为:预变性阶段94℃,60秒;变性阶段98℃,10秒;退火阶段56℃,15秒;延伸阶段72℃,1min/1kb;冷却4℃,10min;变性,退火,延伸三个阶段循环34次,即完成扩增。
得到的PCR产物经AgeI-HF及BsrGI-HF双酶切后跑TAE胶回收。
酶切体系20μL,由PCR产物1μg,10x CutSmart Buffer 2μL,AgeI-HF 1μL,BsrGI-HF 1μL,补水至总体积为20μL;37℃酶切1小时。
酶切完成后加入1x DNA上样缓冲液,于1%的琼脂糖凝胶中电泳160V,30分钟,后于紫外分析仪上分析,找到目的条带,并用手术刀片切下目的条带所在区域的琼脂糖,胶回收DNA片段。
同时,使用本实验室已有的pCSC-IRES-GFP(如图6所示)作为载体backbone,使用AgeI-HF及BsrGI-HF双酶切后,跑TAE胶,切下含载体backbone的核酸胶,胶回收载体片段。与前述纯化后的PCR产物片段通过T4连接酶连接,连接后的产物转化至大肠杆菌Stbl3中。酶切体系与上述PCR产物的酶切体系相同。连接酶连接体系20μL,由回收的PCR产物片段150ng,回收的酶切后载体backbone 50ng,T4连接酶1μL,10x连接缓冲液2μL,补水至总体积为20μL。
转化至大肠杆菌后,将LB平板置于37℃温箱孵育过夜16-18小时;
次日,将LB平板上长出的单克隆用tip头挑出至LB液体培养基(加入0.1mg/ml氨苄青霉素)中于37℃摇床16-18小时。
抽取质粒并Sanger测序验证,得到的pCSC-HGSNAT-WT::MYC::FLAG载体如图7所示。
1.2突变载体的构建
依托上一步构建的pCSC-HGSNAT-WT::MYC::FLAG载体,开展定点诱变(Site-Directed Mutagenesis)。具体地,使用pCSC-HGSNAT-WT::MYC::FLAG载体为模板,分别设计突变引物,扩增全长的HGSNAT::MYC::FLAG载体。设计突变引物对为:
第一组引物对,用于扩增突变p.R344C
上游引物:0584-F:TTGTCTTGGGACAAGGTGTGCATTCCTGGTGTGCTGC(SEQ ID NO:15)
下游引物:0584-R:GCAGCACACCAGGAATGCACACCTTGTCCCAAGACAA(SEQ ID NO:16)
第二组引物对,用于扩增突变p.G248E
上游引物:0585-F:GCGTGGACACCTTCAGGGAGATTGCTCTTATACTCAT(SEQ ID NO:17)
下游引物:0585-R:ATGAGTATAAGAGCAATCTCCCTGAAGGTGTCCACGC(SEQ ID NO:18)
第三组引物,用于扩增突变p.P237Q
见SEQ ID NO 15~20。
上游引物:0586-F:GGCGTCTATCTGCCCTGCAGCCCCGCCTCCGCAGCGT(SEQ ID NO:19)
下游引物:0586-R:ACGCTGCGGAGGCGGGGCTGCAGGGCAGATAGACGCC(SEQ ID NO:20)
扩增相关步骤如下:100ng模板载体加入到50μL反应体系中,反应体系由正向引物和反向引物(10μM)各1.25μL,5 x PrimeSTAR Buffer(Mg2+plus)10μL,dNTP(2.5μM)4μL,PrimeSTAR HS DNA聚合酶(2.5units/μL)0.5μL,补水使总体积为50μL。PCR反应程序为:预变性阶段94℃,60秒;变性阶段98℃,10秒;退火阶段56℃,15秒;延伸阶段72℃,1min/1kb;冷却4℃,10min;变性,退火,延伸三个阶段循环32次,即完成扩增。
得到的PCR产物经DpnI酶切甲基化质粒模板后,转化至大肠杆菌,于次日挑取单菌落扩增,经Sanger测序验证突变成功,分别得到R344C、G248E及P237Q表达载体(见图8~10)。DpnI酶消化体系60μL,由扩增后产物45μL,10 x CutSmart Buffer 6μL,DpnI 0.5μL,补水至总体积为20μL;37℃酶切3小时,于80℃灭活20分钟。
1.3 HGSNAT酶活检测
日常培养293T细胞,完全培养基为DMEM+10% FBS+1%青链霉素双抗。将构建的pCSC-HGSNAT::MYC::FLAG载体(WT、R344C、G248E)各1μg使用PEI转染到293T细胞中。转染过程为:提前传代293T细胞,以3x 105细胞接种到6孔板中。次日,把1μg质粒与无血清的DMEM混合,后添加3倍体积的PEI混匀,室温静置15分钟。混合物缓缓加入培养液中,轻轻摇匀,37℃温箱置12-16小时。转染过夜后,用PBS清洗一次,更换新鲜的10% DMEM完全培养基继续培养。转染48小时后,吸弃培养基,用PBS清洗一遍。加水裂解细胞,收集到EP管中,后使用超声破碎制备细胞裂解液备用。同时用BCA法测定裂解液蛋白浓度。
蛋白裂解液首先使用聚丙烯酰胺凝胶电泳(SDS-PAGE)实验检测,各取30μg蛋白,在10%凝胶中电泳(浓缩胶80V电压跑30分钟;分离胶120V电压跑60~90分钟)。将蛋白电转至PVDF膜上,使用5%的脱脂牛奶封闭抗原,之后分别孵育一抗(MYC、GAPDH)于4℃过夜。次日,去除一抗,使用TBST溶液洗3次,每次洗15分钟。加入HRP偶联的二抗,室温下孵育1小时。TBST洗3次,每次15分钟。最后加入ECL底物发光液,在化学发光仪(Bio-Rad ChemiDocXRS+)中检测蛋白信号。
检测结果见图11,野生型与突变型均可检测出MYC条带,表明均已表达HGSNAT::MYC::FLAG融合蛋白。
在96孔板中,各取10μL蛋白裂解液,加入5μL McIlvain缓冲液(pH 5.5),5μl 3mM底物(4-甲基伞酮-β-D半乳糖苷(MU-βGlcNH2)或者4-甲基蛋白酚对乙酰-β-D氨基葡萄糖(MU-βGlcNAc)),5μL 5mM乙酰辅酶A。于37℃孵育1小时。加入225μL 0.4M甘氨酸缓冲液(pH10.4)。反应液置于酶标仪中,检测激发光波长为360nm、发射光波长为450nm。利用4-甲基伞酮(4-MU)测定标准曲线。计算酶活公式为:
荧光值x 0.25ml x1x 1=nmol/h/mg
斜率0.001ml 1小时蛋白浓度(mg/ml)
酶活性测定结果见图12,由图12可知,R344C、G248E突变的HGSNAT酶活性与野生型相比,分别降至6.0%和4.1%,说明突变后的HGSNAT几乎没有酶活性。同时,突变的HGSNAT没有造成其下游的NAGase的酶活变化,提示突变是特异性导致HGSNAT酶活丧失的。
2、HGSNAT的亚细胞定位检测
将构建的pCSC-HGSNAT::MYC::FLAG载体(WT、R344C、G248E、P237Q)以及溶酶体定位的报告载体pLAMP1::mCherry(addgene 45147)(见图13)使用PEI转染到293T细胞中。转染过程为:提前传代293T细胞,以4x104细胞接种到24孔板的细胞爬片上。次日,把0.25μgHGSNAT质粒和0.25μg报告质粒与无血清的DMEM混合,后添加3倍体积的PEI混匀,室温静置15分钟。混合物缓缓加入培养液中,轻轻摇匀,37℃温箱置12-16小时。转染过夜后,用PBS清洗一次,更换新鲜的10% DMEM完全培养基继续培养。转染48小时后,吸弃培养基,用PBS清洗一遍。使用4%PFA于室温固定细胞20分钟,后置于PBS中备用。
吸弃PBS,用含3% BSA和0.3% Triton-X-100封闭液封闭30分钟。去除封闭液,加一抗(FLAG)于4℃孵育过夜。第二天,用PBST洗3次,每次洗10分钟。加入荧光二抗Alexafluor 488,室温下避光孵育1小时。PBST洗3次,每次10分钟。最后加入Hoechst 33342染核液染色5分钟,用PBS洗5分钟,并使用4% PVA封闭玻片。后于荧光显微镜(Leica DMi8)下观察HGSNAT蛋白在溶酶体中的定位情况。
免疫荧光染色结果见图14,由图14可知,野生型和多态型P237Q的HGSNAT蛋白与溶酶体表达的mCherry在细胞内共定位(信号叠加),而R344C、G248E突变的HGSNAT与溶酶体表达的mCherry在细胞内不完全共定位(信号不叠加),结果说明R344C、G248E突变导致了HGSNAT在溶酶体内定位失败。
值得指出的是,发明人首次发现了单基因遗传病MPSIIIC的致病基因HGSNAT中的新致病突变,发明人以MPSIIIC患病家系为研究对象,对该家系中的患病个体(先证者)进行了全外显子组测序,后经Sanger测序验证先证者与家系中非患病个体;在先证者HGSNAT基因上检测到复合杂合突变:变异1:c.1030C>T(p.Arg344Cys)及变异2:c.743G>A(p.Gly248Glu)。变异1为已报道变异,位于11号外显子的错义突变,该突变造成HGSNAT蛋白344位的精氨酸Arg突变为半胱氨酸Cys;变异2为新发现变异,位于7号外显子的错义突变,该突变造成HGSNAT蛋白248位的甘氨酸Gly突变为谷氨酸Glu。该致病突变也是首次在中国人群发现,通过实施例中提供的检测试剂也在患病家族系中得到验证;此外,还分别通过生物信息学和实验验证了该突变致病性,验证了两个变异导致HGSNAT的活性缺失以及溶酶体定位失败,由此证实两个变异均为有害致病变异,并探讨了相应致病机理。该致病突变,不仅扩展了MPSIIIC致病基因突变谱,同时为开展基因诊断提供了新数据,为该疾病的诊断提供了新的分子生物学基础。
最后有必要在此说明的是:以上实施例只用于对本发明的技术方案作进一步详细地说明,不能理解为对本发明保护范围的限制,任何熟悉本专业的技术人员,在不脱离本申请技术方案的范围内,利用上述揭示的技术内容做出些许的改变、修饰、替代、组合、简化均应为等效置换方式,都包含在本发明的保护范围之内。
Claims (9)
1.黏多糖贮积症IIIC型的致病突变基因,其特征在于:所述致病突变基因为突变的与野生型HGSNAT基因,所述致病突变基因的第7外显子含有一个错义突变位点c.743G>A,所述野生型HGSNAT基因的gDNA序列在NCBI中的Gene ID:138050,所述野生型HGSNAT基因的cDNA在NCBI中的Gene ID:CCDS47852.1。
2.根据权利要求1所述的致病突变基因,其特征在于:第743位核苷酸由G突变为A。
3.根据权利要求1所述的致病突变基因,其特征在于:所述基因的蛋白第248位氨基酸由甘氨酸(Gly)为谷氨酸(Glu)。
4.一种核苷酸,其特征在于:所述核苷酸与MPSIIIC相关,所述核苷酸与野生型HGSNAT基因相比,存在c.743G>A;或突变的HGSNAT基因核苷酸第7外显子含有一个错义突变位点c.743G>A。
5.根据权利要求4所述的核苷酸,其特征在于:所述核苷酸序列的第743位核苷酸由G突变为A。
6.根据权利要求4所述的核苷酸,其特征在于:所述核苷酸序列如SEQ ID NO:7所示。
7.一种蛋白,其特征在于:所述蛋白与MPSIIIC相关,所述蛋白与野生型HGSNAT蛋白相比,第248位氨基酸由甘氨酸(Gly)为谷氨酸(Glu),所述野生型HGSNAT蛋白氨基酸序列如Seq ID:9所示。
8.根据权利要求7所述的蛋白,其特征在于:所述蛋白氨基酸序列如SEQ ID NO:11所示。
9.权利要求1所述的致病突变基因或权利要求4所述的核苷酸或权利要求7所述的蛋白用于制备MPSIIIC检测试剂或检测设备中的应用。
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Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013120018A1 (en) * | 2012-02-09 | 2013-08-15 | Population Diagnostics, Inc. | Methods and compositions for screening and treating developmental disorders |
CA2996445A1 (en) * | 2014-09-05 | 2016-03-10 | Eli Hatchwell | Methods and compositions for inhibiting and treating neurological conditions |
CN109402132A (zh) * | 2018-11-29 | 2019-03-01 | 福州福瑞医学检验实验室有限公司 | 一种编码scn1a基因突变体的核酸及其应用 |
WO2019183630A2 (en) * | 2018-03-23 | 2019-09-26 | The Trustees Of Columbia University In The City Of New York | Gene editing for autosomal dominant diseases |
CN110423805A (zh) * | 2019-08-13 | 2019-11-08 | 上海千贝医疗科技有限公司 | 用于新生儿黏多糖贮积症基因分型的多重pcr引物***及其用途 |
CN110904128A (zh) * | 2019-11-25 | 2020-03-24 | 福州福瑞医学检验实验室有限公司 | 一种编码tek基因突变体的核酸及其应用 |
CN112226440A (zh) * | 2020-11-03 | 2021-01-15 | 南京医科大学 | 一种遗传性原发***的致病突变及其检测试剂 |
CN112626192A (zh) * | 2020-12-16 | 2021-04-09 | 雷博 | 一种基因芯片、包括其的试剂盒及其应用 |
CN114196736A (zh) * | 2021-11-05 | 2022-03-18 | 上海源赏生物科技有限公司 | 一种同步检测多种出生缺陷遗传病全染色体基因分型芯片及其方法和应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8945847B2 (en) * | 2010-05-24 | 2015-02-03 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Methods and kits for ascertaining biosafety of an agent |
-
2023
- 2023-02-20 CN CN202310135848.4A patent/CN116083458B/zh active Active
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013120018A1 (en) * | 2012-02-09 | 2013-08-15 | Population Diagnostics, Inc. | Methods and compositions for screening and treating developmental disorders |
CA2996445A1 (en) * | 2014-09-05 | 2016-03-10 | Eli Hatchwell | Methods and compositions for inhibiting and treating neurological conditions |
WO2019183630A2 (en) * | 2018-03-23 | 2019-09-26 | The Trustees Of Columbia University In The City Of New York | Gene editing for autosomal dominant diseases |
CN109402132A (zh) * | 2018-11-29 | 2019-03-01 | 福州福瑞医学检验实验室有限公司 | 一种编码scn1a基因突变体的核酸及其应用 |
CN110423805A (zh) * | 2019-08-13 | 2019-11-08 | 上海千贝医疗科技有限公司 | 用于新生儿黏多糖贮积症基因分型的多重pcr引物***及其用途 |
CN110904128A (zh) * | 2019-11-25 | 2020-03-24 | 福州福瑞医学检验实验室有限公司 | 一种编码tek基因突变体的核酸及其应用 |
CN112226440A (zh) * | 2020-11-03 | 2021-01-15 | 南京医科大学 | 一种遗传性原发***的致病突变及其检测试剂 |
CN112626192A (zh) * | 2020-12-16 | 2021-04-09 | 雷博 | 一种基因芯片、包括其的试剂盒及其应用 |
CN114196736A (zh) * | 2021-11-05 | 2022-03-18 | 上海源赏生物科技有限公司 | 一种同步检测多种出生缺陷遗传病全染色体基因分型芯片及其方法和应用 |
Non-Patent Citations (3)
Title |
---|
Phenotype prediction of nonsynonymous single nucleotide polymorphisms in human phase II drug/xenobiotic metabolizing enzymes: perspectives on molecular evolution;Science China(Life Sciences);20101005(10);109-113 * |
人类Ⅱ相药物毒物代谢酶非同义单核苷酸多态性的表型预测:分子进化观;郝大程;肖培根;陈士林;;中国科学:生命科学;20110215(02);7-12 * |
粘多糖贮积症基于GAG累积的检测方法;苑晓舟;段晋燕;孟岩;王成彬;;临床检验杂志(电子版);20151230(04);56-62 * |
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