CN114107473A - 一种通过rs6540718来特异性检测少肌症的试剂盒 - Google Patents
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Abstract
本发明公开了一种通过检测rs6540718来特异性检测少肌症的试剂盒。该试剂盒包括检测rs6540718号SNP位点的特异性引物对及特异性荧光探针对、用于荧光定量PCR检测的常规组件等。本发明的试剂盒通过同时检测与少肌症遗传易感性密切相关的rs6540718上的单核苷酸多态性位点基因型来评估个体少肌症遗传易感性。
Description
技术领域
本发明涉及基因工程技术领域中SNP分型检测领域,具体涉及一种通过检测rs6540718来特异性检测少肌症的试剂盒。
背景技术
少肌症又叫肌肉衰减综合症(Sarcopenia),是一种随年龄增加,以骨骼肌肌纤维质量下降、肌肉力量减小、肌肉耐力及代谢能力下降、结缔组织和脂肪增多等为主要特征的老年慢性代谢性疾病。少肌症往往使老年人的活动能力降低,行走、坐立、登高和提重物等日常动作受影响,逐渐“手无缚鸡之力”。肌肉功能下降,导致老年人意外摔伤的几率升高40%。据统计,50%死于意外的老人,其死因是因为摔倒。少肌症老人患行动不便的风险是同龄老人的2-5倍,严重影响老人的生活质量和自理能力。
少肌症除增加残疾和丧失生活自理能力的风险外,还会促使骨质疏松、关节炎等疾病的发展,它同时也是诱发高血压、糖尿病、高血脂等老年疾病的重要原因。少肌症已成为全球残疾和死亡的主要原因之一。在70岁以下的老年人中有13%-24%患有少肌症,在80岁以上的人群中患病率高达50%甚至更高。与少肌症相关的年度经济损失超过了1000亿美元。
过去有关候选基因的大量研究已经表明,很多基因与人类肌肉量的变化相关。与人体瘦体重指数(LMI)相关的40%以上的基因变异可以产生性状差异。随着分子遗传学的进展,人们发现少肌症是多基因遗传病,许多国家都在开展少肌症的遗传关联研究。至今为止至少已通过分子遗传学手段检出了20个人类基因或染色体区域并被报道与少肌症相关。此外,遗传学和分子流行病学的证据均表明遗传因子涉及决定在特定饮食或用药情况下增加或损失肌肉量的易感性,以及在少肌症患者中发生其它相关疾病的高危险性。目前候选基因已经很多,但是绝大部分都在检测准确性上有待于进一步的提高。
至于检测少肌症基因的方法,目前常规的检测方法包括限制性片段长度多态性法(Restriction Fragment Length Polymorphism,RFLP),其原理是当变异影响到某一限制性内切酶的酶切位点时,简单地通过酶切处理患者的PCR产物继而电泳检测是否有变异的方法。但是这种方法存在耗时长,操作繁琐,准确度不高等缺点。也有利用PCR结合DNA测序方法进行基因多态性位点的检测,但是这种方法在大规模人群筛查或检测多个基因多个位点的应用受到一定限制。因此有必要建立一种高通量、高效能、低成本的少肌症易感基因SNP分型方法,以实现临床快速检测或规模化人群筛查。
发明内容
在本发明提供了一种通过检测rs6540718来特异性检测少肌症的试剂盒。
本发明的目的之一是提供一种少肌症易感基因SNP分子标记的筛选方法,所述方法为通过英国生物样本库的大规模遗传队列样本的基因组序列以及通过生物电阻抗法测量的四肢肌肉含量,发现了分子标记位点rs6540718,位置在1号染色体212274413,所述突变为A/C与人的肌肉含量直接相关。
本发明根据NCBI上公开的rs6540718分子标记序列提供一种1q32.3染色体上rs6540718分子标记的扩增引物对,所述引物对的上游引物:GGCAAAGAGAATAGTTCCCC;下游引物:CTAGGAATAAATAGATTTCT;扩增片段大小:261 bp。同时提供了一对检测引物对,基因型一荧光探针序列:5’ -FAM- AGCGGAAAGAaATTGCCAAGC - TAMRA -3’;基因型二荧光探针序列:5’ -VIC- AGCGGAAAGAcATTGCCAAGC - TAMRA -3’。
染色体1q32.3上的rs6540718 SNP检测芯片可用于单独或并行检测染色体1q32.3区段A/C的突变。所述检测芯片采用本领域常规的构建方法制备获得。
本发明提供一种检测少肌症遗传易感性的试剂盒。该试剂盒包括:
检测rs6540718 SNP多态性基因型的特异性引物对SEQ ID NO:2和3所示;PCR反应组件(包括Taq酶、dNTP混合液、MgCl2溶液、反应缓冲液、去离子水等)。
本发明的主要优点
本发明从染色体1q32.3上鉴定获得rs6540718 SNP标记可以用于鉴定人少肌症症状,所述鉴定具有准确性好,特异性高的优点。
本发明的检测方法步骤简单,SNP位点检测可通过一步PCR即可完成,含有SNP位点的目标序列的扩增,避免了反复多次PCR等复杂操作过程中存在的诸多不确定因素,因而可大大提高检测准确率,体现了精确的同时定性、定量分析特征。
具体实施方式
实施例1 SNP分子标记的获得
1,样本
本发明所用样本来自于英国生物样本库(UKB)队列样本。该队列是一个大型前瞻性研究队列,包含50万名来自英国全国各地介于48-73岁的参与者。我们采用一系列严格的纳排标准。排除标准包括自报告性别与遗传性别不一致、性染色体不是整倍型、杂合度过高、基因型缺失率过高等。共有48.7万参与者,排除3.7万参与者后剩余450 243个参与者可供分析,其中男性205 513人,女性244 730人。
2,表型及其建模
体成分包括肌肉含量和脂肪含量使用生物电阻抗法测量。先后测量胳膊和腿部的脂肪含量以及肌肉含量,四肢肌肉含量由胳膊和腿部的肌肉含量相加而得。男性和女性分开建模。在每个性别中,使用四肢脂肪量、年龄、年龄平方、前十个遗传主成分、测量所在地以及基因分型所用芯片作为协变量校正原始四肢肌量。校正后的残差根据逆分位数法转化为标准正态分布并用于下游分析。
3,基因型及其质控
所有参与者均使用定制的UKB基因分型芯片分型,共在常染色体上分型获得784256个单核苷酸多态性位点(SNP)的基因型信息。使用基因型补缺的统计方法补缺到大型基因组参考面板上。该面板由UK10K单体型、千人基因组3期数据以及单体型参考样本组成。共补缺了约九千二百万个SNP。作为纳排标准,排除次等位基因频率<0.1%以及补缺精度<0.3的SNP。经过质控后,共有一千九百万个SNP用于下游分析。
4,遗传关联分析
在每个性别组内,使用BOLT-LMM软件开展混合线性模型的遗传关联分析。在完成两个性别的关联分析后,使用METAL软件的逆方差加权固定效应模型对两个性别的遗传关联信号开展meta分析。全基因组显著性水平设为α=5×10-9。达到该显著性水平的SNP位点同时还要满足性别特异的显著性水平α=5×10-5。也就是说,显著的关联信号不仅meta分析p值<5×10-9,同时在两个性别组内的p值分别<5×10-5。
SNP在两性别组的效应大小使用双侧z检验来比较。鉴定到的SNP位点使用VariantEffect Predictor (VEP)软件进行注释。
5,年龄效应
作为一个典型的老年性疾病,少肌症的遗传效应可能会随着年龄增大而变化。因此,本专利还评估SNP效应随年龄的变化趋势。具体做法是把UKB样本分为6个年龄段:45岁以下(N=54608)、46-50(N=58865)、51-55(N=70253)、56-60(N=89479)、61-65(N=109696)以及66岁以上(N=67342)。每个年龄段内使用BOLT-LMM软件开展遗传关联分析。生成的回归系数对各组别的平均年龄进行meta回归分析。回归分析的P值作为显著性的判断标准。
6,结论
在UKB样本中鉴定出了SNP位点rs6540718。它在男性样本中的效应值是 0.07,女性样本中的效应值是0.04,总体效应值是0.05。效应值在男女组中没有区别。rs6540718的三种基因型AA、CC和AC所对应的四肢肌肉含量平均分别为22.3千克、18.5千克和20.1千克,相应于每个等位基因C减少约2千克。
它的主要遗传关联结果如表1
表1,rs6540718的遗传关联结果
注:等位基因A/C中C是起作用的等位基因。
该位点是一个错义突变,位于1号染色体DTL基因的第14个外显子上。突变导致编码蛋白的第694号氨基酸由赖氨酸突变成苏氨酸。生物信息学分析显示,该突变对蛋白功能有严重的破坏作用。
为了进一步确定rs6540718的遗传效应是否会随着年龄的改变而改变,本专利还进行了年龄特异性分析。分析结果未发现年龄特异性的效应。
综上,错义突变rs6540718的两个等位基因位点A和C能够很好地区分肌肉含量高和低的组。当等位基因为C时,表现为具有少肌症特性,当为A时不表现出少肌症特性。等位基因型CC导致了肌肉含量的降低,从而最终导致少肌症的发生,这种降低在统计学上是极端显著,因此可以通过检测该位点基因型来检测少肌症。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出,对于本领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也将落入本发明权利要求的保护范围内。
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Claims (7)
1.一种检测少肌症的试剂盒,其特征在于:所述试剂盒包括能特异性检测与少肌症相关的SNP位点的探针和引物。
2.如权利要求1所述的试剂盒,所述试剂盒包括能特异性检测rs6540718的探针和引物。
3.如权利要求2所述的试剂盒,其特征在于:所述试剂盒中包括SEQ ID NO:2-5所述的引物或探针序列。
4.SEQ ID NO:2-5所述的引物或探针序列组在制备用于少肌症检测的试剂盒中 的应用。
5.rs6540718做为检测少肌症的靶点的用途。
6.rs6540718在制备用于少肌症检测的试剂盒中的应用。
7.一种检测少肌症的方法,包括使用权利要求1-3任一项所述的试剂盒。
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