【作者】 黄海花；

【导师】 苏敏；

【作者基本信息】 汕头大学 ， 病理学， 2008， 博士

【摘要】 背景位于中国沿海的潮汕及福建闽南地区和位于北方内陆的太行山区,地理位置上相距较远,生活环境迥异,却同为我国的食管癌(Esophageal cancer,EC)高发区,而且具有相似的恶性肿瘤谱。据历史记载,现在潮汕人群的祖先大多为中原内陆居民(河南和山西)途径福建省迁移而来的。语言,家谱,碑文及考古发现也支持这一史实,因此本文提出一假设即潮汕食管癌高危人群与其它两个高危人群具有相似的遗传背景,他们可能来源于一较近的共同祖先,正是由于这种相近的遗传结构,导致了他们共同的食管癌易感性。目的获取太行山区、福建闽南及潮汕地区食管癌高危人群和潮汕地区食管癌人群的Y染色体DNA多态性信息,为本文的假设提供来自父系的遗传学证据。同时为这三个地区人群的个体识别提供参考资料,丰富这些地区的Y染色体单核苷酸多态性(single nucleotide polymorphism, SNP)及短串联重复序列(short tandem repeat, STR)数据库。方法1.选取16个具有东亚人群特异性的Y-SNP及7个Y-STR位点进行研究。用三种方法进行分型,对没有长度变化的SNP标记,用限制性片段长度多态性(Restriction Fragment Length Polymorphism, RFLP)检测;对有长度变化的SNP及7个STR位点先进行荧光PCR扩增,再上基因分析仪进行电泳分型;对于M1,可直接利用琼脂糖凝胶电泳分型。2.根据每个个体所有SNP分型结果及Y染色体国际命名委员会的命名原则,确定每个个体的单体群,分析它们在四个研究人群中的频率分布并进行卡方检验。直接计算SNP基因型及单体群多态性,并进行主成分分析、相关分析及分层聚类分析。3.根据每个个体7个Y-STR的分型结果,计算STR基因型及单体型频率、基因型及单体型多态性。与比对资料一起进行主成分、相关及分层聚类分析,利用Arlequin3.0软件计算群体间的遗传距离,然后再用Mega2.1软件构建邻接法(Neighbor-joining Method,NJ)系统发育树。绘制O2a*及O3e1单体群的网络结构图。结果1. O3*、O3e*及O3e1*单体群均具有M122突变,它们的合计频率在太行山区、福建及潮汕地区食管癌高危人群中分别为60.42%、66.22%及65.16%,是三个高危人群的主要单体群,卡方检验提示它们间的频率差别无统计学意义。潮汕地区食管癌人群这三个单体群的频率也较高,合计频率为36.67%,与三个高危人群不同的是它同时具有较高频率的O2a*单体群(30.0%)。2.抽取四个研究人群及东亚5个不同语系人群主成分分析的前两个主成分值,构建主成分散点图,结果显示三个高危人群均位于图的最右侧,即汉藏人群分布最密集的地方。相关分析发现正是汉藏人群的特征单体群O3e*与汉藏人群的分布趋势相吻合。3. Y-SNP及Y-STR主成分及分层聚类分析显示三个高危人群及潮汕地区食管癌人群聚在一起,相关分析表明三个食管癌高危人群之间呈显著正相关,另外潮汕地区食管癌与太行山区食管癌高危人群间也呈显著正相关关系。4.遗传距离分析显示太行山区、福建及潮汕食管癌高危人群间关系较近,这种亲缘关系在N-J邻接树中得到进一步的反映。5. O2a*单体群网络结构图示,与东亚其他人群比较,本研究的18个O2a*个体紧密聚在一起,位于网络图外围的一个分支上。O3e1单体群网络结构图示在中心节点及次中心节点均有四个人群的个体分布,它们具有相同的Y-STR突变。6.除四个研究人群的DYS391及潮汕食管癌人群的DYS389I位点的基因多态性小于0.5外,四个人群在其它Y-STR位点的基因多样性均大于0.5。另外四个人群7个Y-STR单体型多样性均在0.980以上。结论1.父系遗传结构研究结果提示河南太行山区、福建及潮汕高危人群均属于典型的汉藏人群,它们之间具有较近的亲缘关系,可能来自一较近的共同祖先。2.潮汕地区食管癌人群与三个食管癌高危人群也存在较近亲缘关系,但其祖先可能与百越人群具有更多的基因交流。食管癌在潮汕高危人群中可能不是随机发生的,O2a*单倍群可能是筛选潮汕食管癌易感人群有用的候选标记。3. 7个Y-STR在四个人群中具有丰富的遗传多态性,由这些位点组成的Y-STR单体型有很高的个体识别率,具有重要的法医学应用价值。更多还原

【Abstract】 BackgroundChaoshan, Fujian and Henan Taihang Mountain EC high-risk areas were geographically distant and furthermore their living environment was very diverse, but they were all famous for their high incidence and mortality rate for EC, and populations among these regions share similar pedigree of malignancy. According to historical records, Han inhabitants of north-central China (Henan and Shanxi Hans) continuously migrated into Chaoshan area via Fujian due to warfare and famines, and gradually became the predominant inhabitants of Chaoshan area, which were confirmed by the records on genealogy, stone tablet and archaeological discovery and the language-speaking. Therefore we hypothesized that these three EC high-risk populations might share common genetic traits and descended from a recent common ancestor.ObjectivesTo obtain the polymorphism information of Y-chromosome DNA on the four studied populations and provide the patrilineal genetic evidence to verify our hypothesis. In the same time, it also could provide the reference data for individual identification in the three study areas and would abundant the Y-chromosome database among these regions。Methods1. 16 SNP loci that were highly informative in East Asians and 7 STR markers were selected to study. Three strategies were introduced to type Y-SNP and Y-STR. For SNPs without length changes, was typed using PCR-RFLP assay. For SNPs with length variation, and Y-STRs, fluorescence PCR was used. The product of PCR was electrophoresed on a 3100 genetic analyzer to type. Genotype of M1 was conducted by agarose gel electrophoresis directly after PCR. Y-SNP haplogroup and Y-STR haplotype assignments were made based on the three typing results.2. The Y-SNP haplogroup of every individual was defined according to the typing results and following the Y-chromosome consortium nomenclature. Their frequencies and diversities were calculated and compared among the three EC high-risk populations and Chaoshan area EC high-risk populations. Chi-square test was performed to show the difference of haplogroup distribution in the studied populations. Meanwhile, the allele diversity values (DP) and the haplotype diversity for each Y-STR locus was directly calculated.3. Principal component analysis and correlation analysis were carried out to show the genetic affinity among the three EC high-risk populations and other groups for comparision. The paternal genetic relationship among the EC high-risk populations was further displayed by hierarchical cluster analysis. The extent of genetic differentiation of the populations was estimated by the Rst statistic on the basis of the Y-STR haplotypes. Neighbor joining tree was constructed according to the Rst distance matrix to show the phylogenic structure among the populations. O2a* and O3e1 haplogroup network was drawn to show the detailed difference and association among the four studied populations and other populations for compared.Results1. O3*, O3e* and O3e1*, sharing M122 mutation, were the predominant haplogroups in the three high-risk populations. The overall frequencies of these three haplogroups in Taihang mountain, Fujian and Chaoshan EC high-risk populations were 60.42%, 66.22% and 65.16%, respectively. X2 test showed no significant difference among them (p>0.05). These three haplogroups were also frequent in Chaoshan area EC population (30.0%), but they were significantly lower than those in the three EC high-risk populations (p<0.05). In addition, haplogroup O2a* was more frequent in Chaoshan area EC population than in EC high-risk populations (p<0.05).2. Principal component analysis was carried out among the populations of all three studied areas and other five language families’populations in East Asia. Using the values of PC1 and PC2 as height values, the principal components plot was drawn. The result showed that those from the three studied areas clustered together in the rightmost part of the PC map, especially the three EC high-risk populations. They all located among Sino-Tibetan population. Correlation analysis displayed that only haplogroup O3e* was consistent with the distribution of Sino-Tibetan populations. 3. Principal component and hierarchical clustering analysis based on the Y-SNP and Y-STR frequencies among populations coming from the three studied areas and other regions of China displayed that the four studied populations formed a distinctive cluster. Correlation analysis showed that haplogroup frequency distribution in the Chaoshan EC high-risk population was significantly positively correlated to those in the Taihang Mountain and Fujian EC high-risk populations. Chaoshan area EC patients were also significantly positively correlated to Taihang Mountain high-risk population.4. Rst values showed a close relationship among Taihang Mountain,Fujian and Chaoshan high-risk population. The close genetic affinity among the three high-risk populations was also displayed by the N-J phylogeny.5. O2a* network analysis showed that compared with other Chinese and East Asia populations, populations in the three studied areas formed a close cluster and distributed at the edge of the network. O3el haplogroup network showed that the O3e1 individuals from the three studied areas shared the same Y-STR mutations in the central and sub-central node of the network.6. The allele diversity values (DP) of these loci was higher than 0.5 except for that of DYS391 in the four studied populations and DYS389II in Chaoshan area EC patients. The haplotype diversity for the seven Y-STRs was all above 0.980.Conclusions1. Taihang Mountain, Fujian and Chaoshan EC high-risk populations were all typical Sino-Tibetan population. They might share similar patrilineal genetic background and descended from a recent common ancestor, which also confirmed that our hypothesis on genetic backgroundd of Chaoshan EC high-risk population was relatively creditable.2. Chaoshan area EC population also showed closer genetic affinity with the three high-risk populations, however, it might have more gene flow between the ancestor of Chaoshan area EC population and the Baiyue populations. Esophageal cancer might not occur randomly in Chaoshan area population. Haplgroups O2a* might be a candidate genetic background marker for screening population susceptible to EC in Chaoshan area.3. The 7 Y-STR loci had high discriminating ability and their haplotypes were highly polymorphic in the three studied area populations. They were suitable for forensic individual identification and paternity testing.更多还原