河南人及广东客家人的G6PD基因和L1CAM基因单倍域的研究

【外文题名】The Haplotype Blocks Of Glucose-6-phosphate Dehydrogenase Gene And L1 cell adhesion molecule Gene From Henan and Guangdong Hakka Populations

【作者】 刘晗

【关键词】 葡萄糖 6 磷酸脱氢酶 单核苷酸多态性 单倍域 连锁不平衡 变性高效液相色谱技术 优势选择 人群迁徙

【外文关键词】 Glucose 6 Phosphate Dehydrogenase SNPs Haplotype block LD DHPLC positive selection population immigration

【导师姓名】蒋玮莹

【学位名称】硕士

【学位年度】2008

【学位授予单位】暂无

【所属分类】Q532,I236.61,O657.7,Q343.1,Q343.2,Q524,B845,I236.65,O613.62,Q554,Q346

【录入时间】2008-01-01

【全文挂接】 万方挂接 读秀挂接

【摘要】、广西、海南、贵州、云南、四川、台湾等省份。由于G6PD缺乏症的发病率与疟疾的地理分布的一致性，引起了人们对G6PD缺乏的红细胞可能具有抗疟性的思考。G6PD缺乏症属X连锁不完全显性遗传，男性半合子G6PD活性呈显著性缺乏，女性杂合子酶活性变化范围较大，可呈显著性降低，也可在正常范围。G6PD基因是一种看家基因，位于Xq28，由13个外显子及12个内含子组成，全长20,114bp。分子生物学水平的分析显示不同种族人群G6PD基因突变类型和发生频率不同。中国人主要以c.1388 GA、c.1376 GT、c.95 AG、c.871GA突变为主。单倍域是若干个相邻SNPs组成的具有高连锁不平衡（LD）的染色体区段，一个单倍域只有少数几种单倍型。只有突变和重组才能打破始祖单倍域的结构。单倍域的研究将为探讨优势选择、不同群体的遗传多态性、疾病与遗传的关联性、致病基因和致病因子、药效及副作用、人类起源进化及迁徙史等提供完整的人类基因组信息和有效的研究工具。形成长距离高LD的原因之一是优势选择作用。优势选择的一个重要特征是等位基因频率在一个相当短的时间内快速上升，并稳定下来。而重组没有显著破坏自然选择作用形成单倍型结构。因此自然优势选择的标志是受优势选择的等位基因在人群中有较高的频率，由这些等位基因参与组成的core haplotype具有不同寻常高的长距离LD。本研究首先采用NBT定性法在河南周口共筛查了2027人（男性1047人，女性980人），发现G6PD缺乏症患者5人（男性4人，女性1人）。然后我们联合应用聚合酶链反应（polymerase chain reaction, PCR）和变性高效液相色谱（denaturing high performance liquid chromatography,DHPLC）及DNA测序技术对200例河南人(男性84人，女性116人)进行G6PD基因分型。河南人中G6PD基因共发现SNPs10种，其中有8种在数据库中已见报道；1种曾在山东人中发现过，但数据库中未见报道；还有1种g.13401CT是首次在中国人中发现的变异位点。在河南人群中我们还在1例筛查阳性的病人中检测到了第七外显子的一个新突变c.682GA，引起了第228位氨基酸天冬氨酸转换成天冬酰胺，但二级结构的分析未发现结构的改变。L1CAM基因也位于Xq28，距离G6PD基因619kb。我们用PCR联合DHPLC和测序技术对200例河南人和59例广东客家G6PD缺乏男性患者L1CAM基因的五个区域进行研究。在客家G6PD缺乏患者组中，发现了一个新的L1CAM基因多态位点g.10382GA；在河南人群中发现了一个新突变c.1721GA引起了第574位甘氨酸转换成丝氨酸。实验结果显示这个区域的SNPs位点少且出现频率也低，提示进化上的保守性。我们分析比较了四组人群的G6PD和L1CAM的SNPs分布、单倍域和单倍型结构。对客家G6PD缺乏症患者在G6PD基因和L1CAM的LD分析初步构建出了一个长639kb的单倍域，已经显示出长的LD趋势，说明中国人常见的G6PD c.1376 GT、c.1388 GA、c.95 A G、c.871 GA等致病性突变受到了优势选择。我们的研究结果还显示了河南人组和山东人组的单倍域和单倍型结构一致，由相同的7个SNPs位点构成。而广东客家阴性对照人组构成的单倍域较小,构成单倍域的 SNPs点只有前者7个中的3个，其结构反映了始祖单倍域的部分遗迹。据历史记载，客家人是在唐朝时期由中原地区迁徙到南方而来的。我们的实验结果与它相符。我们提出了一种新的研究人类起源和迁徙的遗传学方法。...

【外文摘要】　 Glucose-6-phosphate dehydrogenase(G6PD)deficiency [MIM:+305900]is the most common human metabolic disorder, affecting more than 400 million people worldwide. The incidence of the disease is higher in tropical zone and subtropic zone. In China, G6PD deficiency distributes mainly in South, especially in Guangdong, Guangxi, Hainan, Guizhou, Yunnan, Sichuan, Hongkong and Taiwan. The prevalence of the G6PD deficiency is correlated with geographic distribution of malaria, which has led to be thought that erythrocytes of G6PD deficient individuals may incur partial protection against malaria infection. Because the G6PD gene is located on the X chromosome, the clinical symptoms of the disease are usually confined to hemizygous males. G6PD gene consists of 13 exons and 12 introns, spanning 20,114bp. The Molecular analysis of G6PD deficiency has revealed that different ethnic groups have different gene variants. The mainly pathogenic mutations in Chinese are G6PD c.1376 GT、c.1388 GA、c.95 A G and c.871 GA.Within the haplotype block, the linkage disequilibrium degree was very high, and only several major haplotypes existed. The ancestry haplotype block structure can only be separated by mutation and recombination. The study of haplotype block will provide integrated human genome information and effective research tool for the studies on positive selection, genetics and disease, drug side effect，the history of human origin and immigration and so on. Positive selection causes an unusually rapid rise in allele frequency, occurring over a short enough time that recombination does not substantially break down the haplotype on which the selected mutation occurs. It is one of the causes to form long distant high LD. A signature of positive natural selection is thus an allele having unusually long-range LD given its population frequency. With informed consent, a total of 2027 individuals (1047 males and 980 females) from Henan were screened for G6PD deficiency by the method named Nitrblue tetrazolium paper strip (NBT) .We found 5 G6PD deficiency patients(4 males and 1 female). Among 2027 individuals, 200 of unrelated samples (84 males and 116 females) were subjected to analyze with PCR, DHPLC and DNA direct sequencing. 10 variable alleles in G6PD gene were detected. 8 of them can be found in the SNP database of GeneBank and HapMap; 1 of them has been found in Shandong population but have not been reported; The remnant g.13401 CT is found firstly in Chinese population. In Henan population we also found a new mutation in a G6PD deficiency patient diagnosed by NBT method. It happened in the 7th exon c682 producing a Asn substituting for. Asp at code 228. No change has been found in the secondary structure analysis. We also scanned 5 regions of the L1CAM gene in 200 Henan samples and 59 Guangdong Hakka G6PD deficiency male patients. L1CAM gene located 619kb from G6PD gene. Our result showed that the SNPs in L1CAM were rare and had lower frequency. We found a new SNP g.10382 GA in the L1CAM gene in Guangdong Hakka，who are G6PD deficiency patients. Additionally, a new mutation L1CAM c.1721GA which result in Gly 574 to Ser substitution was found in Henan population. We analyzed the distribution of SNPs, haplotype and haplotype block in 4 groups. The result showed that the haplotype blocks from Henan and Shandong populations are the same, which are composed of 7 identical SNPs, whereas the one acting as the control from Guangdong Hakka population is different from them .The haplotype block from Hakka who are G6PD normality was formed by 3 SNPs, which are included in the 7 SNPs mentioned above. According to the annals, the Hakka population migrated to southern China, including Xingning city, from Henan, Shandong, Shanxi and so on. Hence, we considered the difference is caused by immigration (gene flow) between populations and the environment effects. Our analysis showed the SNPs of the G6PD deficient patients from Guangdong Hakka population have formed a very long haplotype block which is 639kb in length .It has initially displayed high LD tendency, illustrated the most popular pathogenic mutations in China including G6PD c.1376 GT、c.1388 GA、c.95 A G and c.871 GA. have been influenced by positive selection. However, considering the limited sample number and the SNPs besides G6PD gene, further work is under going....