【作者】 冯洁；

【导师】 杨泽；

【作者基本信息】 中国协和医科大学 ， 遗传学， 2010， 博士

【摘要】 长寿是一个受遗传因素影响的复杂性状,也是健康老人的标志。近年来研究证明衰老和寿命延长受基因控制。这些基因多半与能量代谢有关,分别位于核和线粒体基因组内,在生命进化过程中高度保守。因此,系统研究核和线粒体基因组变异对老年长寿的影响,更好的了解长寿的原因和机制,对于降低老年性疾病的发病风险和开展一级预防,从而保障老年人的健康是非常重要的。方法1)经生物信息学分析,确定了线粒体基因组和核内APOE基因变异为拟定的长寿关联候选基因。2)通过收集长寿家族遗传信息和长寿表型信息,并且通过case-control设计,收集长寿老人和无长寿家族史、当地一般人群的对照组。3)长寿相关表型分析：与对照组比较其相应的生理或生化测定指标和临床疾病相关的体征信息和长寿表型特征。4)长寿关联基因研究：采用群体1筛查,群体2,3验证的策略。4.1)经线粒体全基因组扫描,建立线粒体基因信息数据库和长寿群体的mtDNA系统发育进化树,并寻找长寿关联的线粒体基因及单倍组。4.2)经核内APOE基因分型,检测长寿关联的APOE基因变异。4.3)多群体验证线粒体基因及单倍组和APOE基因与长寿的关联,并进行META分析证明。4.4)探索APOE基因与线粒体基因的交互作用。5)分析长寿关联基因型对表型的影响。6)初步开展长寿关联线粒体基因变异的功能研究。7)采用多种遗传统计学软件,进行模型分析。结果1)本研究中共收集814个长寿老人(年龄≥90岁)和1136个无长寿家族史、当地一般人群的对照组(年龄30-65岁),男女比例为1：2.8,其中,巴马壮族群体共1012人(2001年274人,2008年738人)和永福汉族群体(2008年)共938人。2)共计筛查45个相关表型,其中长寿老人与对照群体相比,有7个表型具统计学差异(SBP, DBP, FBG, TC, TG, LDL-C和BMI)。3)经线粒体全基因组测序,在巴马长寿老人中发现了225个突变(mtSNPs),并根据测序结果发现了25个线粒体单倍组。4)在2008年巴马群体中发现：单倍组F与长寿关联,在长寿组和对照组的分布频率存在明显统计学差异(21.0% vs 13.5%,p=0.01),单倍组F有利于长寿(OR:1.703,95%CI:1.153-2.514)。5)进一步分析发现单倍组F中的3个mtSNPs(m.397C＞T, m.13928G>C, m.10310G>A)与长寿长寿关联(p=0.009, OR：1.602,95%CI:1.134-2.262; p=0.021, OR:1.507,95%CI:1.075-2.114和p=0.01, OR:1.703,95%CI:1.153-2.514)。6)经与Mitomap中rCRS序列比较,发现m.13928G＞C非同义突变引起MT-ND5蛋白的一个氨基酸的改变(Ser531Thr)。进一步进行MT-ND5蛋白二级结构预测和疏水性分析显示这个氨基酸改变Ser531Thr位于第7和第8个跨膜螺旋的loop结构中,并使得ND5蛋白局部亲水性增高。7)经构建mtDNA系统发育进化树,显示巴马长寿群体属于中国南方群体。8)经核内基因组中的APOE基因分型,在2001年巴马群体中检验APOE基因变异与长寿的关联,显示长寿老人中APOEε3/ε3基因型频率(78.9%)明显高于对照人群(67.8%),并且具有统计学差异(p=0.04)；而长寿老人中APOEε3/ε4基因型频率和APOEε4等位基因频率明显低于对照群体,存在统计学差异(5.5% vs 13.7%,p=0.02和4.6% vs 10.6%,p=0.01)。9)在2008年巴马群体和2008年永福群体中验证APOE基因变异与长寿的关联,2008年巴马群体的分析结果显示长寿老人中APOEe4等位基因频率和APOEε3/ε4基因型频率明显低于对照群体,与长寿存在负关联(p=0.004, OR:0.565,95%CI:0.380-0.838和p=0.044, OR:0.626,95%CI:0.396-0.991);而APOEs3等位基因型频率和ε3／ε3基因型频率明显高于对照群体,与长寿正关联(p=0.008, OR:1.427,95%CI:1.095-1.859和p=0.007, OR:1.530,95%CI:1.124-2.083)。2008年永福群体的分析结果显示在长寿老人中APOEε4等位基因和APOEs3/ε4基因型频率明显低于对照群体,与长寿负关联(p<0.01, OR:0.29,95%CI:0.18-0.49和p<0.01, OR:0.32,95%CI:0.18-0.58),而APOEε3等位基因型和APOEε3/ε3基因型频率明显高于对照群体,与长寿正关联(p<0.01,OR:2.06,95%CI:1.52-2.78和p＜0.01,OR:2.16,95%CI:1.55-3.02).10)经多个群体Meta分析表明ε4等位基因携带者(ε3／ε4和ε4／ε4基因型总和)是影响长寿的风险因素,与长寿负关联(合并OR=0.42,95%CI：0.36-0.49)。ε3／ε3基因型是影响长寿的保护因素,与长寿正关联(合并OR=1.47,95%CI:1.25-1.74)。11)经交互作用分析mtDNA和APOE基因变异对长寿的影响,发现在APOEε4携带者中长寿老人单倍组F分布频率明显高于对照组的分布频率(28.9%vs 8.8%, p=0.010, OR:4.237,95%C1:1.335-13.446)。12)基因型-表型分析了mtDNA基因变异及APOEs4基因对长寿相关表型的影响。发现在长寿老人中单倍组F, m.13928C和m.10310G与高密度脂蛋白增高相关(p=0.03,p=0.04和p=0.03),且m.13928C与总胆固醇水平降低相关(p=0.04)。显示在长寿老人中APOEε4携带者比非APOEε4携带者的总胆固醇和低密度脂蛋白水平高(p=0.003 and p=0.012),而APOEε3/ε3携带者比未携带APOEε3/ε3的长寿老人的高密度脂蛋白水平高,有统计学差异(p=0.005)。结论1)发现mtDNA单倍组F与长寿关联,可能是长寿的保护因素。2)发现了3个mtSNPs(m.3970C＞T, m.13928G>C, m.10310G>A)与长寿关联；其中非同义突变m.13928G>C (Ser531Thr)在功能上可能起重要的保护作用。3)2个群体证实APOEε4基因与长寿关联,可能是长寿的风险因素。4) mtDNA单倍组F与APOEε4基因可能存在有交互作用。5)提示着mtDNA基因变异和APOE基因变异可能影响长寿老人的健康表型,如血浆总胆固醇,低密度脂蛋白和高密度脂蛋白的水平。更多还原

【Abstract】 Longevity is a complex trait with genetic inheritance, and a marker for healthy and successful aging. Recent studies demonstrated that the lifespan and aging is regulated by genes. Most of these genes are involved in energy metabolism as well as mostly highly conservative and located in nuclear and mitochondrial genome. Therefore, the systematic study of the role of nDNA and mtDNA genetic variants in healthy longevity and successful aging is very important to better understand the reason and mechanism of health longevity and to reduce the risk of aged-related diseases and improve the life quality of the elders.Objectives:To explore’the role of nDNA and mtDNA genetic variants in healthy longevity and successful aging, we analyzed the association of nDNA and mtDNA genetic variants with healthy longevity and successful aging, and employed a preliminary study on the function mechanism of nDNA and mtDNA genetic variants.Methods:1) By bioinformatics analysis, the mitochondrial genome and nuclear APOE gene are selected as the candidate genes associated with longevity.2) Through collecting the information of familial longevity and longevity-related phenotype and by case-control design, long-lived individuals who were 90 years old or above was selected as case sample and the local general population whose parents didn’t survived to 90 years old or above was selected as control sample.3) Longevity-related phenotype analysis:the physiological or biochemical measurement index and longevity-related phenotype were compared between long-lived individuals and controls.4) The study of genes associated with longevity:identifying genes associated with longevity in population 1, and replicating genes associated with longevity in population 2 and 3.4.1) By sequencing whole mitochondrial genome, construct mtDNA phylogeny and identify mtDNA genetic variants associated with longevity.4.2) By genotyping APOE gene variants, detecting APOE gene variants associated with longevity in population 1.4.3) Replicating APOE gene variants associated with longevity in population 2 and 3, and conducting Meta-analysis in multi-populatipns.4.4) Exploring the interaction between APOE gene and mitochondrial genetics variants.5) Analyzing the effect of genes associated with longevity on the longevity-related phenotype.6) A preliminary study on the function of the mtDNA genetic variant associated with longevity.7) By a variety of statistical software packages, conducing genetic model analysis. Results:1)A total of 814"longevity" cases were classified as participants who had survived to age 90 years or more, with a total of 1136 "younger controls" less than 65 years of age, including 1012 Bama individuals(247 individuals in 2001 and 738 individuals in 2008) and 938 Yongfu individuals(in 2008). The ratio of female and male is 2.8:1.2) A total of 43 phenotypes were analyzed in this study. There were statistical significance for 7 phenotypes (SBP, DBP, FBG, TC, TG, LDL-C and BMI) between long-lived population and controls.3) By sequencing the whole mitochondrial genome, 225 mtSNPs and 25 mtDNA haplogroups were found in 20 long-lived individuals.4) Haplogroup F was found to be significantly more frequent in longevity cases (21.0%) than in local controls (13.5%) in Bama population in 2008. There were significantly statistical significance for haplogroup F between two groups (p=0.013), conferring approximately 1.7 times of the protective effect (OR:1.703,95%CI:1.153-2.514). 5)The three mtSNPs in haplogroup F m.3970C＞T, m.13928G>C, m.10310G>A) were found to be consistently associated with longevity(p=0.009, OR:1.602,95%CI: 1.134-2.262; p=0.021, OR:1.507,95%CI:1.075-2.114和1 p=0.01, OR:1.703,95%CI: 1.153-2.514).6) By the comparative analysis of rCRS sequence, the mutation m.13928G/C causes an amino acid change Ser531Thr in the MT-ND5 protein. According to the protein secondary-structure and hydrophilicity analysis, the Ser531Thr polymorphism is located in the loop structure between the 7th and 8th transmembrane helices and increases local hydrophilicity in the loop structure.7) The mtDNA phylogeny constructed showed long-lived populations could have originated from ancestor in South China.8) Compared to control subjects in Bama population in 2001, the homozygousε3/ε3 genotype frequency in long-lived population was significantly higher (78.9% versus 67.8%, p=0.04), whereas the heterozygousε3/ε4 genotype andε4 allele frequency were significantly lower (5.5%versus 13.7%, p=0.02, and 4.6%versus 10.6%, p=0.01). 9) Further replicate the association of APOE gene with longevity in Bama population in 2008 and Yongfu population in 2008. Results in Bama population in 2008 showed the APOEε3 allele and the APOE s3/s3 genotype in long-lived individuals is higher frequent than those in control subjects, positively associated with longevity (p=0.008, OR:1.427, 95%CI:1.095-1.859 and p=0.007, OR:1.530,95%CI:1.124-2.083), whereas APOEε4 allele and the APOEε3/ε4 genotype were significantly lower frequent than those in controls, negatively associated with longevity (p=0.004, OR:0.565,95%Cl:0.380-0.838 and p=0.044, OR:0.626,95%CI:0.396-0.991). Results in Yongfu population in 2008 indicated the APOEε3 allele and the APOEε3/ε3 genotype in long-lived individuals is higher frequent than those in control subjects, positively associated with longevity (p<0.01, OR:2.06,95%CI:1.52-2.78 and p<0.01,OR:2.16,95%CI:1.55-3.02), whereas APOEε4 allele and the APOEε3/e4 genotype were significantly lower frequent than those in controls, negatively associated with longevity (p<0.01,OR:0.29,95%CI: 0.18-0.49 and p<0.01, OR:0.32,95%CI:0.18-0.58).10) The results of meta-analysis in 14 populations suggestedε4-carriers (the s3/s4 andε4/ε4 genotypes) were positively associated with longevity and the APOE s3/s3 genotype was negatively associated with longevity. The combined ORs for APOEε4-carriers was OR=0.42,95%CI:0.36-0.49 and the combined ORs for APOEε3/ε3 genotype OR=1.47,95%CI:1.25-1.74.11) The effect of the interaction of mtDNA genetic variants and APOE gene on longevity was analyzed, suggesting among APOEε4-carriers, mtDNA haplogroup F was higher frequent in long-lived population than that in controls (28.9%vs 8.8%, p=0.010, OR:4.237, 95%CI:1.335-13.446).12) The relationship of FBG, TG, TC, HDL-C and LDL-C levels with mtDNA variants was tested. The results showed that there was a significant relation of TC and HDL-C levels to mtDNA variants. Haplogroup F, m.13928C and m.10310A were associated with markedly higher HDL-C levels, and m.13928C was associated with lower TC levels in long-lived cases. APOEε4-carrier was associated with significantly higher TC and LDL-C levels (p=0.003 and p=0.012), and APOEε3/s3 was associated with higher HDL-C levels (p=0.005) in long-lived individuals.Conclusions:1) The mtDNA haplogroup F was found to be associated with healthy longevity, conferring the protective effect.2) The three mtSNPs (mt3970C>T, mt13928G>C and mt10310G>A) were found to be associated with longevity, and an amino acid changes Ser531Thr (mt13928G/C) in the MT-ND5 gene might account for the beneficial effect of F.3) it was confirmed APOE gene associated with longevity in multi-population APOEε4 gene increased the risk effect for successful aging and longevity, whereas APOEε3 gene conferred protective effect for healthy longevity.4) There could be the interaction of haplogroup F and APOE s4 gene.5) The mtDNA and APOE genetic variants possibly influence healthy phenotype of long-lived individuals, such as TC, LDL-C and HDL.更多还原