摘要
随着“人类基因组计划”(human genome project,HGP)的完成,人类已了解自己基因组的全部核苷酸序列,在全面寻找功能基因的同时,“人类基因组计划”的另一个重要目标是阐明在特定基因组区域内的序列差异。单核苷酸多态性(Single nucleotide polymorphisms,SNPs),作为基因组中最普遍的序列差异,在生物医学、遗传学等诸多领域,如复杂性疾病病因研究、药物敏感性研究以及人群进化史研究中发挥着重要的作用。
酪氨酸羟化酶(Tyrosine hydroxylase,TH)作为体内的一种重要神经递质儿茶酚胺合成的限速酶,由于mRNA编码的核酸序列的差异,TH基因表现出基因的多态性。近年来TH的基因多态性在法医学研究领域中受到了广泛的关注。首先精神疾病作为在司法精神病学研究中占首位的鉴定案例,众多的学者们从不同的角度对其遗传特征进行了广泛的研究,研究结果表明精神疾病的发生具有一定的遗传性,这种遗传性可能与TH基因的突变密切相关。其次有学者提出TH基因多态性在一定程度上反映了个体的差异度,因此这种基因多态性有可能在法医学个人识别和亲子鉴定上具有潜在的应用前景。但以往由于实验手段的限制,尚缺乏对TH基因进行全面系统的分析。目前随着一些高灵敏度、高通量的基因分型技术的日趋成熟,采用新的方法以基因组为模板广泛分析TH基因的突变成为可能,这无疑将会为该基因在今后法医学的应用及疾病的相关性研究等方面提供无法比拟的先决条件。
目的
本研究采用DNA测序和实时荧光定量PCR(Realtime PCR)技术,选择存在于人类TH基因外显子3和内含子9上的两个SNP位点进行调查,针对辽宁地区的精神病患者及部分家系,通过分析与精神疾病密切相关的脑神经递质TH基因的多态性,并与正常群体进行比较,探讨这一易感基因与精神疾病的相关性以及其在人类遗传学中的意义。同时通过对TH基因位点SNP的高通量系统分析,计算获得的有关法医学应用参数,为其在法医学中的应用奠定基础。
材料与方法
1、样品
1.1中国北方汉族127例无血缘关系健康个体静脉抗凝血由中国医科大学法医学院法医血清学教研室提供。
1.2 173例尼泊尔国藏族人DNA样品由日本国久留米大学医学部法医人类遗传学教室友好提供。
1.3 130例日本福冈地区健康日本人DNA样品由日本国久留米大学医学部法医人类遗传学教室友好提供。
1.4 133例居住在中国北方的患有精神疾病的血液样品,以及7例患有精神疾患的家系血液样品均由辽宁开原精神病医院提供。
1.5 73例居住在中国北方的抑郁症患者的DNA样品由中国医科大学心理学教研室友好提供。
2实验方法
2.1模板DNA制备采用酚/氯仿法抽提DNA。
2.2 PCR扩增目的DNA片段:
G334A引物序列:F:5'-CAGCGCGGCTCCAGCCCGT-3',R:5'-AGTCCCGGGCGGACAGCAGGC-3';
C5162G引物序列:F:5'-CAGTGCACCCAGTATATCCG-3',R:5'-CCGTCGCACCCCCCACCCTC-3'。
2.3 DNA测序:测序PCR反应应用BigDye Terminator v3.1 Cycle SequencingKit和ABI 3130全自动序列测定分析仪。测序用引物序列:TH-exon10-L:5'-CAGCATGGGCACGTGCCCCA-3',TH-exon9-U:5'-CGCGTGGCCCCTTGCAGAGC-3'。
2.4统计学处理方法
由试验所得的数据依照参考文献计算出基因型频率、等位基因频率和杂合度,同时与实际调查所获得的基因型频率之间进行检验,验证是否符Hardy-Weinberg定律。以上数据均利用SPSS统计软件包进行计算分析。
结果
1、人类TH基因突变的命名
本研究在分析人NT 009237的基因组Contig的同时,确定出了TH的基因组结构。研究结果显示所分析鉴定的两个突变点分别定位于外显子3中的第7个碱基对(G/A)和内含子9中的第8个碱基对(C/G),依据美国立生物技术信息中心对SNP的命名方法,分别命名为G334A(Thr314Met)和rs12419447(C/G)或C5162G。
2、G334A和C5162G两位点的群体学分布数据
在G334A位点,三个调查的群体中均存在CC、CT和TT三种基因型。两种常见的等位基因频率为C=日本人＞中国北方汉族＞尼泊尔藏族(即0.269＞0.214＞0.205),T=尼泊尔藏族＞中国北方汉族＞日本人(即0.795＞0.786＞0.731)。在C5162G位点,三个调查的群体中均存在CC和CG两种基因型,但均没有检出GG基因型,说明后一种基因型在东亚及靠近东亚的群体中可能相当罕见。两种常见的等位基因频率为C=日本人＞中国北方汉族＞尼泊尔藏族(即0.977＞0.961＞0.922),G=尼泊尔藏族＞中国北方汉族＞日本人(即0.078＞0.039＞0.023)。
3、G334A和C5162G两位点数据的法医学意义
个人识别机率(Probability of discrimination power,DP)在G334A位点上为日本人＞中国北方汉族＞尼泊尔藏族(即0.5593＞0.5012＞0.4916);非父排除率(Excluding probability of paternity,EPP)为日本人＞中国北方汉族＞尼泊尔藏族(即0.1580＞0.1399＞0.1364);杂合度(Heterozygosity,H)为中国北方汉族＞日本人＞尼泊尔藏族(即0.3629＞0.3538＞0.3509)。在C5162G位点上DP值为尼泊尔藏族＞中国北方汉族＞日本人(即0.2633＞0.1455＞0.0878);EPP值为尼泊尔藏族＞中国北方汉族＞日本人(即0.0667＞0.0361＞0.0220);H值为尼泊尔藏族＞中国北方汉族＞日本人(即0.1561＞0.0787＞0.0462)。
4、G334A位点在精神疾病和抑郁症患者中的分布
中国北方地区精神疾患群体G334A位点的基因频率分布为:C=0.133,T=0.867;忧郁症群体的基因频率分布为:C=0.116,T=0.884。
5、C5162G位点在精神疾病和抑郁症患者中的分布
中国北方地区精神疾患群体C5162G位点的基因频率分布为:C=0.962,G=0.038;忧郁症群体C5162G位点的基因频率分布为:C=0.959,G=0.041。
6、人类TH基因SNP在患精神疾病家系中的基因型分布
TH基因G334A位点,6名子代个体及5例双亲均为T/T基因型;另两例组合中,父亲也为T/T基因型,而母亲为C/T基因型,两名子代的基因型为C/T。C5162G位点,7名子代个体及6例双亲均为C/C基因型;另一例组合中,父亲也为C/C基因型,而母亲为C/G基因型,子代的基因型为C/G。
7、家系的疾病表现及与TH基因SNP的相关性
7名患病子代个体的父亲无精神疾病史,而所有母亲均先前被诊断为精神病患者,对应的TH基因G334A和C5162G两位点的基因型在6名先证者均为T/T-C/C组合,只有1名先证者的两位点基因型组合为T/T-C/G;患有精神疾患的母亲中5人的两位点基因型组合也为T/T-C/C,另两人分别为C/T-C/C和C/T-C/G基因型组合。
结论
1、本研究利用互联网资源及相关报道数据阐述了人类TH基因的基因组结构,详细给出了外显子及内含子序列碱基数目,并对有关TH基因中的SNP提出了一种命名法。
2、首次报道了人类TH基因外显子3中的G334A和内含子9中的C5162G二位点在中国北方汉族、尼泊尔地区藏族和日本福冈地区日本人三个群体中的等位基因频率分布,为其在法医学个人识别和亲子鉴定中的应用提供了有必需的基础数据。
3、应用TaqMan荧光实时定量PCR技术报道了人类TH基因外显子3中的G334A和内含子9中的C5162G两基因座在精神疾患及抑郁症两个群体中的等位基因频率分布,为遗传流行病学的研究提供了重要的基础数据。
4、本研究对G334A和C5162G两基因座基因型与精神疾病家系表型进行了比较,发现先证者的两个基因座大多表现出与亲代患有精神疾病的个体同样的基因型。
5、比较正常人群与精神疾患和忧郁症群体的G334A和C5162G两基因座基因型频率的分布,发现G334A基因座基因型的分布在正常人群与精神疾患和忧郁症群体之间皆存在显著差异;而C5162G基因座基因型的分布正常人群与精神疾患和忧郁症群体之间无显著差异。
Preface
Since we have finished the human gene project(HGP),we have known about all the gene order.We are studying the functional gene,as well as finding the differences among the specific gene order.Single nucleotide polymorphisms(SNPs) as the most comon differences play an important part in the research of disease,medicine,and evolution.
Tyrosine hydroxylase(TH) as an important rate limiting enzyme in the human body.It had a gene polymorphism caused by the differences among mRNA coding. More attention has been paid to the TH polymorphism for forensic research in these years.It has been proved that the mental disorders had some heritage by many research. And the heritage had some connections with the TH polymorphism.Some other researchers thought that the TH polymorphism was different among the different people,so there may be an important signification in telling different people.It may be useful to individual identification and paternity test.For the limiting of the technology, we have no systemic research results.With the development of technology,TH polymorphism research had become more and more popular.
Objective
The aim of this study is to evaluate the relationship between the TH gene and mental disorder by comparing the two TH gene SNP points(extron 3 and intron 9) of patients with mental disorder and those of human without mental disorder.And the significance of TH gene polymorphology in human genetic and in forensic medicine is also discussed.
Materials and Methods
1、specimen
1.1 Normal contract group:127 cases all come from blood donors in Shenyang blood center with no Schizophrenia or Schizophrenia family history or suicide disease. They are all Han nationality.
1.2 173 tibetans' blood from Nepal offered by JAPAN university.
1.3 130 normal japaneses' blood offered by JAPAN university.
1.4 133 cases with chizophrenia or Schizophrenia,and 7 Schizophrenia family history offered by Kaiyuan madhouse.
1.5 73 cases with depression offered by china medical university.
2、methods
2.1 Template DNA is made by hydroxybenzene/chloroform method from venous anti -thrombin DNA.
2.2 Use PCR to amplify the target DNA fragment:
Inducer sequence of G334A:F:5'-CAGCGCGGCTCCAGCCCGT-3',R:5'-AGT -CCCGGGCGGACAGCAGGC-3';Inducer sequence of C5162G:F:5'-CAGTGCA -CCCAGTATATCCG-3',R:5'-CCGTCGCACCCCCCACCCTC-3'。
2.3 DNA sequencing:we use BigDye Terminator v.3.1 Cycle Sequencing Kit and ABI 3130 automatic sequencing machine.primer are TH-exon10-L: 5'-CAGCATGGG- CACGTGCCCCA-3',TH-exon9-U: 5'-CGCGTGGCCCCTTGCAGAGC-3'.
2.4 Statistic analysis:Record all individual classification result;calculate each allele and gene type frequency;perform Hardy-Weinberg balance test..Using spss software.
Results
1、Naming of gene mutation of human TH.
We have make the order of TH gene,which named G334A(Thr314Met) and rs12419447(C/G) or C5162G.
2、G334A and C5162G distribution data.
We found CC,CT,and TT in all three groups.Most common allele frequency is C=Japanese＞north chinese＞tibetan in nepal(0.269＞0.214＞0.205);T=tibetan in nepal＞north chinese＞Japanese(0.795＞0.786＞0.731).In C5162G there are CC and CG,but no GG,which proves that the GG is very rare.Most common allele frequency is C=Japanese＞north chinese＞tibetan in nepal(0.977＞0.961＞0.922);G=tibetan in nepal＞north chinese＞Japanese(0.078＞0.039＞0.023).
3、G334A and C5162G signifitance in forensic medicine.
Probability of discrimination power(DP):Japanese＞north chinese＞tibetan in nepal(0.5593＞0.5012＞0.4916);Excluding probability of paternity(EPP):Japanese＞north chinese＞tibetan in nepal(0.1580＞0.1399＞0.1364);Heterozygosity,H:north chinese＞Japanese＞tibetan in nepal(0.3629＞0.3538＞0.3509).In C5162G,DP:tibetan in nepal＞north chinese＞Japanese(0.2633＞0.1455＞0.0878);EPP:tibetan in nepal＞north chinese＞Japanese(0.0667＞0.0361＞0.0220);H:tibetan in nepal＞north chinese＞Japanese(0.1561＞0.0787＞0.0462).
4、G334A distribution In north china:C=0.133,T=0.867 in Schizoph- renia,and C=0.116,T=0.884 in depression.
5、C5162G distribution:C=0.962,G=0.038 in Schizophrenia,and C=0.959,G=0.041 in depression.
6、SNPs of TH in the Schizophrenia family.
For G334A spot,6 filial generations and 5 parents are all T/T gene type,and in the other two cases,father is T/T,mother is C/T,both filial generations are C/T;for C5162G spot,7 filial generations and 6 parents are all C/C gene type,and in the other case,father is C/C,mother is C/G,filial generation is C/G.
7、The relationship between familial disease and the SNPs of TH.
Any of the fathers is the families had no Schizophrenia history,and all the mothers had Schizophrenia history.In G334A and C5162G spots of the TH only one is T/T-C/G,the other 6 cases are T/T-C/C.The five mothers with Schizophrenia is T/T-C/C,and the other two cases is C/T-C/C and C/T-C/G.
Conclusion
1、Depending on the data from the internet,we discript the gene order of TH of human being,and a name method.
2、First report G334A and C5162G.Describe the gene type distribution in north chinese,Japanese,and tibetan in nepal.
3、Depending on the TaqMan real time PCR technology,we determinate G334A and C5162G gene distribution in Schizophrenia and depression,which will be very useful to genetic research.
4、Compare G334A and C5162G in the mental disease.We find that prohand usually is the same gene type with the parents with the mental disease.
5、Compare G334A and C5162G between normal people and the patients with mental disease.Finding that in G334A there are significant difference between normal people and patients with mental disease,and there are no significant difference in C5162G.
引文
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