CN1884528A - 与利培酮临床疗效相关的核酸序列 - Google Patents
与利培酮临床疗效相关的核酸序列 Download PDFInfo
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Abstract
本发明涉及的是一种基因技术领域的与利培酮临床疗效相关的核酸序列。本发明所分离出的DNA分子，它具有SEQ ID NO：1所示的序列，在SEQ ID NO：1所示序列的第251位为单核苷酸多态性位点rs1799978，所述的单核苷酸多态性位点rs1799978是指在SEQ NO：1所示序列中的第251为存在A。所述的DNA，在其互补链上为T等位位点，位于DRD2基因上游－241位。所述的SEQ ID NO：1序列，其中，第251位的单核苷酸多态性位点为A纯合子。通过本发明预测利培酮的临床疗效不依赖于医生的主观判断，且时效性强，因此是临床合理用药可以借助的有力工具。
Description
技术领域
本发明涉及的是一种基因技术领域的核酸序列，特别是一种与利培酮临床疗效相关的核酸序列。
背景技术
利培酮(risperidone)是新一代的抗精神病药物，1984年由比利时杨森制药公司开发，商品名为维思通(Risperidal)，为苯异噁唑(benzisoxazole)衍生物，它与5-HT2受体和DRD2受体都有很高的亲和力，与第一代抗精神病药物相比，利培酮对精神***症的阳性症状及阴性症状均有良好疗效，且锥体外系症状(EPS)等不良反应发生率低(Leysen JE，Gommeren W，Eens A，De Chaffog De CoucellesD，Stoof JC，Janssen PAJ.Biochemical profile of risperidone，a newantipsychotic.J Pharmacol Exp Ther 1988；247：661-670)。在实际应用中，利培酮的临床疗效存在很大的个体差异，仅有约50％的用药患者可获得较好疗效，其它的服药患者则疗效不明显、甚至出现严重的不良反应，而目前临床上除了试错试验外，尚无有效预测利培酮药物效应的方法，这种临床疗效的不确定性已成为用药安全的重要隐患，而且浪费了大量医疗资源(Miyamoto S，Duncan GE，Marx CE，Lieberman JA.Treatments for schizophrenia：acritical review of pharmacology and mechanisms of action of antipsychoticdrugs.Mol Psychiatry 2005；10(1)：79-104)。现有研究结果显示，遗传因素是利培酮临床疗效产生个体差异的重要原因，与利培酮临床疗效相关的基因主要包括代谢酶、转运体、靶点等的编码基因。DRD2是利培酮的主要作用靶点(Kapur S，Zipursky RB，Remington G.Clinical and theoretical implicationsof 5-HT2 and D2 receptor occupancy of clozapine，risperidone，andolanzapine in schizophrenia.Am J Psychiatry 1999；156(2)：286-93)，DRD2基因的多态性可显著影响DRD2的功能，这提示DRD2基因的多态性可能与利培酮临床疗效的个体差异有关。
单核苷酸多态性rs1799978(A-241G)位于DRD2基因上游调控区，经对现有技术文献的检索发现，至今未见关于DRD2基因的单核苷酸多态性位点rs1799978与利培酮临床疗效相关，可作为分子标记用于预测利培酮临床疗效的相关报道。
发明内容
本发明的目的在于克服现有技术中的不足，提供一种与利培酮临床疗效相关的核酸序列。使其可用于预测利培酮的临床疗效、指导利培酮的个体化治疗。
本发明是通过以下技术方案实现的，本发明所分离出的DNA分子，它具有SEQ ID NO：1所示的序列，在SEQ ID NO：1所示序列的第251位为单核苷酸多态性位点rs1799978，所述的单核苷酸多态性位点rs1799978，是指在SEQ NO：1所示序列中的第251为存在A，在其DNA互补链上为T等位位点，位于DRD2基因上游-241位。
所述的SEQ ID NO：1序列，其中，第251位的单核苷酸多态性位点为A纯合子。个体利用利培酮治疗更易于获得较好疗效。根据该多态性的等位位点特征可以预测利培酮的临床疗效，指导临床合理用药。
本发明所述利培酮临床疗效相关核酸序列如SEQ NO.1所示：
(a)序列特征：
*长度：1000碱基对
*类型：核酸
*链型：双链
*拓扑结构：线形
(b)分子类型：基因组DNA(gDNA文库)
(c)假设：否
(d)反义：否
(e)最初来源：人
(f)SEQ ID NO.1核酸序列描述如下：
ggcctccttt tgcatttcca tgtcaggtca actccaaaca ggctttttcc 50
tttggatgag tccagcaata gaactgacta catagattcc aagacctgaa 100
gtcagaaaac ggatgcggac ctcttccaac acctcctctc cttgcctgac 150
ttgctggatg ggtggtgcct cccgcaaccc ttggcttctg agtcctcaaa 200
ggagaagact ggcgagcaga cggtgaggac ccagcctgca atcacagctt 250
attactctgg gtgtgggtgg gagcgcagtg ggcggtcgag ggttgcgttc 300
ccgcctcaaa acaagggatg gcggaatccc ccaacccctc ctacccgttc 350
aggccgggga tcgccgagga ggtacagctc ctttggtggg gggcgggggc 400
ggggcctgtc tcaggggcgg ggaccggggc acctccctcc cgcgctcccc 450
gcgctcgggc gccgcagagc tgtccagctt cagtgccgaa ccggcagcct 500
cacgcgcgca ccgcgccgcc tccgccccgt ccccgcgctc cctcctgccc 550
gcccgccccg cgcccggccc cgccccgccc cgccccgccg cggcccgtcc 600
actgctcccc gcgggccaga gccggccgag ctgctgcccg ccggggctct 650
gaacggcgcg gcggggccgg gagccaggga ccggccgagg agagtggcgg 700
ccccggacgg ctgccggagg ggcggccgcg cgtggatgcg gcgggagctg 750
gaagcctcaa gcagccggcg ccgtctctgc ccccgggcgc cctatggctt 800
gaaggtaagc accggccgga tggagcggcg ggcgactcag tgtcacgggg 850
agaggaggag ggacctccaa ggggagcgag gccaggacca gaagtttgtg 900
cggccactcc tggccgcctg ctcgggagcg cccgcctggg atccggctct 950
gcatgggctc cccgtcgggc gggggcagag ggcgtctgga gcccgcggct 1000
序列中251号的字母代表单核苷酸多态性位点(single nucleotidepolymorphisms，SNP)A/G的等位位点位置。
利用本发明提供的利培酮临床疗效相关的单核苷酸多态性位点的核酸序列，构建预测利培酮临床疗效的试剂盒，可用于利培酮的个体化治疗。
具体实施方式
以下结合具体实施例，以进一步说明本发明。应理解，以下实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1：基因的收集和抽提
精神***症患者来自于中国上海市精神卫生中心，以DSM-IV(中国精神病分类方案与诊断标准第二版修订版)为诊断标准。在知情同意的前提下参加本课题的研究，采集血液，并签署知情同意书。所有入组患者均无第二代抗精神病药物用药史，经过周期为4周的药物清洗期后开始临床实验。分别于实验开始、4周、8周时采用简明精神病量表(Brief Psychiatric Rating Scale，BPRS)对患者临床症状评分，BPRS减分率大于或等于40％者定义为有效，小于40％者为无效。
实施例2：核苷酸序列的获得以及单核苷酸多态性位点rs1799978的检测
本发明采用直接测序的方法对DRD2基因单核苷酸多态性位点rs1799978(其等位位点为A/G)等位位点在利培酮治疗有效患者组和利培酮治疗无效患者组中的频率分布情况进行了检测。PCR反应均采用96孔板，用Gene Amp PCR system9700完成。
(1)PCR反应
扩增引物：
正向引物(Pri-F)：5’-CCCCACCAAAGGAGCTGTACCT-3’(SEQ ID NO：2)
反向引物(Pri-R)：5’-CAGAAAACGGATGCGGACCTCT-3’(SEQ ID NO：3)
反应试剂：
反应体系总体积为10.0ul，其中
ddH2O 5.8ul 灭菌双蒸水
Buffer 1.0ul 聚合反应缓冲液包含Mg2+ Tris-HCl溶液
dNTP 1.0ul 2mM dNTP溶液
Pri-F 0.5ul 10pmol/ul正向引物
Pri-R 0.5ul 10pmol/ul反向引物
Taq 0.2ul DNA合成多聚酶
DNA 1.0ul 10ng/ul人体DNA扩增模板
反应条件：
94℃ 4min，37×(94℃ 30s，68℃ 30s，72℃ 60s)，72℃ 10min，4℃∞
(2)测序反应
PCR产物采用shrimp alkaline phosphatase(Roche，Basel，Switzerland)and exonulease I纯化，具体过程按说明书操作。
测序反应体系：
DNA 3.0ul 纯化后DNA，计200ng
BDT 1.0ul ABI BigDyeTerminator Cylce sequencing Ready Reaction
Kit 3.1
Primer 1.0ul所获得的PCR产物的正向或反向引物3pmol/ul
测序反应条件：
96℃ 2min，40×(94℃ 30s，50℃ 30s，60℃ 120s)，4℃∞
结束后加入70％乙醇避光静置15分钟，4000转/分钟离心30分钟，甩去乙醇，70％乙醇洗两次，静置晾干，加入HIDI变性后上样。测序采用ABI PRISM3100 DNA Sequencer进行。
实施例3：单核苷酸多态性位点rs1799978与利培酮临床疗效的相关性
统计：利用SPSS10.0计算等位位点、基因型的分布和Hardy-Weinberg平衡，并计算Odds Ratio(OR)及其95％的可信区间，采用G Power Program计算所用样本量的统计学力度，选用卡方检验进行统计学分析，统计学的显著性水平设定为P小于0.05。
结果：位于11q22-23区域DRD2基因上游调控区的rs1799978(其等位位点为A/G)等位位点在利培酮治疗有效患者组和利培酮治疗无效患者组中的频率分布情况见表1。
表1.rs1799978等位位点和基因型在利培酮治疗有效患者组和无效患者组中的分布和分析结果
| 分组 | 样本数 | 基因型(频率％) | 等位位点数(频率％) |
| 无效组有效组POR(95％CI) | 5372 | AA AG GG27(50.94) 20(37.74) 6(11.32)52(72.22) 17(23.61) 3(4.17)0.040 | A G74(69.81) 32(30.19)121(84.03) 23(15.97)0.0070.440(0.239-0.808) |
注：OR：Odds Ratio；CI：Confidence intervals
从上表可见，位于11q22-23区域DRD2基因上的常见型单核苷酸多态性位点rs1799978的A等位位点，即在其DNA互补链上为T等位位点，在利培酮治疗有效组中的分布频率明显大于其在利培酮治疗无效组中的频率(P＝0.007)，因此可作为预测利培酮临床疗效的标记分子。rs1799978位于DRD2基因上游-241位，可能影响DRD2基因的表达，或与其它功能性多态性位点连锁。
本发明的具有实用性的例证：
1)可以利用本发明所介绍的rs1799978的A等位位点(即在其DNA互补链上为T等位位点)作为预测利培酮临床疗效的标记分子，为临床合理用药提供依据。
2)利用本发明提供的多态性位点rs1799978的核酸序列，构建用于指导利培酮个体化治疗的试剂盒。
3)由于DRD2基因是多种抗精神病药物的作用靶点，因此，本发明为今后对DRD2基因与其他抗精神病药物疗效关系公开了前期最宝贵的经验、实验基础和应用技术。
序列表
<110>上海交通大学
<120>与利培酮临床疗效相关的核酸序列
<160>1
<210>1
<211>1000
<212>DNA
<213>人类(Homo sapiens)
<220>
<221>allele
<222>(251)…(251)
<223>序列中第251位核苷酸为a或g
<400>1
ggcctccttt tgcatttcca tgtcaggtca actccaaaca ggctttttcc 50
tttggatgag tccagcaata gaactgacta catagattcc aagacctgaa 100
gtcagaaaac ggatgcggac ctcttccaac acctcctctc cttgcctgac 150
ttgctggatg ggtggtgcct cccgcaaccc ttggcttctg agtcctcaaa 200
ggagaagact ggcgagcaga cggtgaggac ccagcctgca atcacagctt 250
attactctgg gtgtgggtgg gagcgcagtg ggcggtcgag ggttgcgttc 300
ccgcctcaaa acaagggatg gcggaatccc ccaacccctc ctacccgttc 350
aggccgggga tcgccgagga ggtacagctc ctttggtggg gggcgggggc 400
ggggcctgtc tcaggggcgg ggaccggggc acctccctcc cgcgctcccc 450
gcgctcgggc gccgcagagc tgtccagctt cagtgccgaa ccggcagcct 500
cacgcgcgca ccgcgccgcc tccgccccgt ccccgcgctc cctcctgccc 550
gcccgccccg cgcccggccc cgccccgccc cgccccgccg cggcccgtcc 600
actgctcccc gcgggccaga gccggccgag ctgctgcccg ccggggctct 650
gaacggcgcg gcggggccgg gagccaggga ccggccgagg agagtggcgg 700
ccccggacgg ctgccggagg ggcggccgcg cgtggatgcg gcgggagctg 750
gaagcctcaa gcagccggcg ccgtctctgc ccccgggcgc cctatggctt 800
gaaggtaagc accggccgga tggagcggcg ggcgactcag tgtcacgggg 850
agaggaggag ggacctccaa ggggagcgag gccaggacca gaagtttgtg 900
cggccactcc tggccgcctg ctcgggagcg cccgcctggg atccggctct 950
gcatgggctc cccgtcgggc gggggcagag ggcgtctgga gcccgcggct 1000
Claims (4)
1、一种与利培酮临床疗效相关的核酸序列，其特征在于，所分离出的DNA分子，它具有SEQ ID NO：1所示的序列，在SEQ ID NO：1所示序列的第251位为单核苷酸多态性位点rs1799978。
2、根据权利要求1所述的与利培酮临床疗效相关的核酸序列，其特征是，所述的单核苷酸多态性位点rs1799978，是指在SEQ NO：1所示序列中的第251为存在A。
3、根据权利要求2所述的与利培酮临床疗效相关的核酸序列，其特征是，所述的DNA，在其互补链上为T等位位点，位于DRD2基因上游-241位。
4、根据权利要求1所述的与利培酮临床疗效相关的核酸序列，其特征是，所述的SEQ ID NO：1序列，其中，第251位的单核苷酸多态性位点为A纯合子。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109777870A (zh) * | 2018-11-07 | 2019-05-21 | 上海康黎医学检验所有限公司 | 一种用于指导人精神疾病用药的试剂盒及其检测方法 |
| CN111440861A (zh) * | 2020-04-01 | 2020-07-24 | 上海市精神卫生中心（上海市心理咨询培训中心） | 甲基化的miR-124基因在制备用于诊断抑郁症的标志物中的用途 |
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2006
- 2006-06-29 CN CNB2006100283294A patent/CN100491528C/zh active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109777870A (zh) * | 2018-11-07 | 2019-05-21 | 上海康黎医学检验所有限公司 | 一种用于指导人精神疾病用药的试剂盒及其检测方法 |
| CN109777870B (zh) * | 2018-11-07 | 2019-10-29 | 上海康黎医学检验所有限公司 | 一种用于指导人精神疾病用药的试剂盒及其检测方法 |
| CN111440861A (zh) * | 2020-04-01 | 2020-07-24 | 上海市精神卫生中心（上海市心理咨询培训中心） | 甲基化的miR-124基因在制备用于诊断抑郁症的标志物中的用途 |
| CN111440861B (zh) * | 2020-04-01 | 2022-11-04 | 上海市精神卫生中心（上海市心理咨询培训中心） | 甲基化的miR-124基因在制备用于诊断抑郁症的标志物中的用途 |
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| CN100491528C (zh) | 2009-05-27 |
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