【作者】 杨康鹃；

【导师】 金元哲；

【作者基本信息】 延边大学 ， 生理学， 2007， 博士

【摘要】 目的2型糖尿病(T2DM)是由于胰岛素抵抗或胰岛素分泌相对不足而导致慢性高血糖的一种常见的具有明显异质性的多基因遗传病，又称为复杂性疾病(complex disease)。遗传因素在T2DM的发生发展中起着重要作用，其发病的遗传特征以多个微效基因(minor gene)和环境因素共同参与其致病过程。本课题旨在分析中国延边的朝鲜族与汉族人群T2DM的发生发展的遗传特征和危险因素，并通过检测T2DM的候选基因过氧化物酶体增殖物激活受体-γ2(proxisome proliferators activated receptor-γ2，PPAR-γ2)、脂联素(adipnectin，αpM-1)和过氧化物酶体增殖物激活受体-γ共激活子1(peroxisome proliferator-activated receptor-γcoactivatorl，PGC-1)基因单核苷酸多态性(single nucleotide polymerphism，SNPs)的单体型及连锁不平衡(linkage disequilibrium，LD)与T2DM相关性分析，以期筛选T2DM的危险因子和单体型，为T2DM的诊断、治疗和预防提供理论依据。同时对中国延边的朝鲜族和汉族两个群体的遗传结构和亲缘关系分析提供遗传学上的证据。方法本课题分三部分实施。1．采用遗传流行病学手段，随机抽取延边地区朝鲜族和汉族222名T2DM患者作为先证者组，收集其一级亲属为1747人；糖耐量正常(NGT)对照组458名其一级亲属为2261人；以问卷形式填写涵盖一般情况、生活习惯和体征，每个研究对象均绘制系谱，共进行27项实验室检查及体格检查；应用阈值理论估算T2DM的遗传率；利用Li-Mantel-Gsrt法进行同胞的分离比比较，分析T2DM的遗传模式；采用单因素和多因素非条件Logistic回归分析法，筛查T2DM的危险因素。2．通过生物信息学方法在NCBI的dbSNP数据库(Human SNPView)及HapMap数据库查找PPAR-γ2、αpM-1和PGC-1基因的SNP位点，获悉数据库中已构建的单体型及单体型标签SNP(haplotype tag SNP，htSNP)，结合文献及研究现状确定本次分析的30个SNPs位点。采用聚合酶链反应-单链构象多态性(PCR-SSCP)技术及直接测序确定基因型，对延边地区散发T2DM患者259例(朝鲜族103人，汉族156人)及对照组221例(朝鲜族108人，汉族113人)进行基因型频率和等位基因频率病例-对照关联研究，采用SHEsis在线软件(http://202.120.7.14/analysis/)进行SNPs的单倍型和连锁不平衡分析，筛查T2DM危险因子。3．利用SNP Assistant version1.0.11(BioData，Ltd)软件在9个T2DM核心家系中进行PPAR-γ2、αpM-1和PGC-1基因8个常见SNPs位点传递不平衡检验(transm-ission-disequilibrium test，TDT)，判断各种SNPs与T2DM的连锁关系。结果1．遗传流行病学研究结果(1)延边地区朝鲜族、汉族T2DM患者的一级亲属的患病率分别为5.09％和4.82％，均高于对照组人群的一级亲属的患病率1.64％和0.89％(P＜0.001)，患病危险度(OR)分别增高3.10倍和4.82倍；其中朝鲜族女性患者的一级亲属的患病率(12.39％)明显高于朝鲜族男性、汉族男性及汉族女性患者的一级亲属的患病率(4.67％、4.85％和4.80％)(P＜0.05)，朝鲜族女性患者的一级亲属患病危险度比其他亲属高2.6倍以上。汉族和朝朝鲜族先证者组一级亲属之间患病率或对照组一级亲属之间患病率均无显著性差异(P=0.056和P=0.809)。(2)延边地区T2DM的总遗传率为39.6％，朝鲜族和汉族的遗传率分别为39.4％和51.6％，汉族高于朝鲜族，但无统计学意义(P＞0.05)；朝鲜族女性一级亲属遗传率(76.4％)显著高于男性一级亲属遗传率(37.2％)(P＜0.001)。(3)T2DM在朝鲜族和汉族同胞中的分离比分别为0.21和0.15，低于0.25，不符合单基因遗传病的特征，支持T2DM多基因遗传模式。(4)在单因素分析基础上进入模型危险因素有糖尿病家族史、腰臀比、高血压、甜食摄入量4个因素与延边地区人群T2DM具有显著性差异(P＜0.05)。2．T2DM候选基因PPAR-γ2、αpM-1和PGC-1的SNPs单体型连锁不平衡分析结果(1)通过对延边地区朝鲜族和汉族T2DM组和糖耐量正常对照组中PPAR-γ2、αpM-1和PGC-1基因部分扫描共检出15个SNPs位点，既PPAR-γ2基因的Pro12Ala(C＞G)和C161T(His477His)；αpM-1基因-11391G＞A、-11377C＞G、-11156insCA、T45G(Gly15Gly)、+276C＞A、+349A＞G、R221S和H241P；PGC-1基因Thr394Thr(G＞A)、IVS2+52C＞A、Gly482Ser(G＞A)、Thr528Thr(G＞A)、Ser577Leu(C＞T)。15个SNP各种基因型频率和等位基因频率符合Hardy-Weinberg平衡条件，达到遗传平衡(P＞0.05)，证明选择的样本具有群体代表。其中等位基因频率低于1％的有4个位点：-11391G＞A(A频率为0.009)、R221S(C＞A)(A频率为0.003)、H241P(A＞C)(C频率为0.0015)、Ser577Ler(C＞T)(T频率为0.0013)。本次研究未发现的15个SNPs：PPAR-γ2基因Pro115Gln(C＞A)、IVS2+40C＞T、Gln438Gln(A＞G)、Ser457Ser(A＞G)、Leu481Leu(C＞T)及αpM-1基因-11426G＞A、-11043C＞T、G162A(Gly54Val)、+1711C＞T、+1795A＞G、+1833C＞A和PGC-1基因Ser74Leu(C＞T)、Leu410Iso(C＞A)、Leu438Ser(T＞C)、Asp475Asp(C＞T)。(2)延边汉族和朝鲜族PPAR-γ2基因Pro12Ala和C161T基因型频率和等位基因频率与T2DM相关性分析结果：①Pro12Ala基因型频率在汉族T2DM组与NGT组分别为PP：0.954和0.960，PA：0.046和0.040，A等位基因频率分别为0.023和0.020，T2DM组与NGT组之间无显著性差异(P＞0.05)；朝鲜族T2DM组与NGT组PP：0.948和0.955，PA：0.052和0.045，A等位基因频率分别为0.026和0.022，T2DM组与NGT组之间也无显著性差异(P＞0.05)；延边地区人群中未发现Ala12Ala纯合子：朝鲜族和汉族各种基因型频率和等位基因频率均无显著性差异(P＞0.05)。②C161T多态的基因型频率在汉族T2DM组与NGT组分别为：CC：0.615和0.655，CT：0.346和0.345，TT：0.039和0；T等位基因频率分别为0.212和0.172，T2DM组与NGT组之间无显著性差异(P＞0．05)：朝鲜族T2DM组与NGT组各种基因型频率分别为：CC：0.684和0.611，CT：0.263和0.333，TT：0.053和0.056，T等位基因频率分别为0.184和0.222，T2DM组与NGT组之间无显著性差异(P＞0.05)；朝鲜族和汉族各种基因型频率和等位基因频率均无显著性差异(P＞0.05)。③Pro12Ala与C161T单倍型和连锁不平衡分析结果显示共存在三个单倍型PC、PT和AC，其中AC单倍型在NGT组和T2DM组频率为0和0.059，存在显著差异(P=0.032)，危险度OR=0.001(95％CI0.000-0.010)，Pro12Ala与C161T为弱连锁不平衡(D’=0.332)。④PPAR-γ2基因Pro12Ala和C161T多态有明显种族差异，但不存在民族或地域差异。(3)αpM-1基因5个SNPs各种基因型频率NGT组与T2DM无显著差异(P＞0.05)，但不排除样本少的误差；各种等位基因频率在T2DM组与NGT组分别为：-11377C＞G的G等位基因频率分别为：0.237与0.225，-11156insCA的发生插入(Ⅰ)等位基因频率分别为0.132和0.158，T45G的G等位基因频率分别为：0.294和0.389，+276G＞T的T等位基因频率分别为：0.294和0289，+349A＞G的G等位基因频率分别为：0.324和0.389。各种等位基因频率在NGT组与T2DM无显著差异(P＞0.05)。(4)延边汉族和朝鲜族PGC-1基因IVS2+52C＞A、Thr394Thr(G＞A)、Gly482Ser(G＞A)、Thr528Thr(G＞A)基因型频率和等位基因频率在NGT组与T2DM相关性分析结果：①4个SNPs的各种基因型频率和等位基因频率分布在朝鲜族和汉族之间无显著性差异(P＞0.05)。②4个SNPs的基因型频率及等位基因频率在NGT组和T2DM组间的分布也无显著性差异(P＞0.05)，结果显示PGC-1基因IVS2+52C＞A、Thr394Thr(G＞A)、Gly482Ser(G＞A)、Thr528Thr(G＞A)单个SNP均与T2DM发生无相关性。③4个SNPs共存在8种常见的单体型：AAAA、AAGG、AGAA、AGGG、CAAA、CAGG、CGAA和CGGG；汉族人群不存在单体型CAAA，朝鲜族不存在单体型AAAA；单体型CAAA仅在朝鲜族NGT组存在，此单体型是否是朝鲜族正常人群特异的单体型的结论，还需扩大样本定论；同时还发现单体型CAGG在汉族T2DM组显著高于NGT组(P=0.007，OR=0.128(95％CI 0.025-0.666)，此结果暗示CAGG可能是汉族的危险单倍型。④4个SNPs两两位点联锁不平衡分析结果显示Gly482Ser与Thr528Thr呈完全连锁不平衡，提示Gly482Ser可作为单倍型的标签SNP，无需再检测Thr528Thr。⑤PGC-1基因Thr394Thr与Gly482Ser联合基因型分析结果显示在NGT组与T2DM组共存在8种联合基因型形式：AA-AA(0与8.8)；AA-GA(0与2.9)；AA-GG(7.7与2.9)；GA-GA(23.1与11.8)；GA-GG(11.5与20.6)；GG-AA(11.5与20.6)；GG-GA(26.9与26.5)；GG-GG(19.2与5.9)；8种联合基型均与T2DM发生无相关性(P＞0.05)。⑥PGC-1基因3个突变热区的SNPs IVS2+52C＞A、Gly482Ser和Thr528Thr多态性与其他种族人群比较存在差异性。⑦IVS2+52C＞A不同基因型与各种生理生化指标分析结果：在汉族的T2DM组携带CA+AA基因型人群BMI显著高于CC基因型人((P＜0.05)，而其他的指标如WHR、FPG、TC、TG、HDL-C及LDL-C在两种基因型之间无显著差异(P＞0.05)：在NGT组携带CC基因型人群FPG明显高于CA+AA基因型人群(P=0.007)，同时CC基因型人群HDL-C明显低于CA+AA基因型人群(P=0.011)，而在其他的指标如BMI、WHR、TC、TG及LDL-C两种基因型之间无显著差异(P＞0.05)。在朝鲜族NGT组携带CC基因型人比携带CA+AA基因型人LDL-C显著增高(P＜0.05)，而其他的指标如BMI、WHR、FPG、TC、TG、HDL-C在两种基因型之间无显著差异(P＞0.05)；在T2DM组各种基因型与BMI、WHR、FPG、TC、TG、HDL-C及LDL-C无显著差异(P＞0.05)。(5)PGC-1基因IVS2+52C＞A和Gly482Ser与PPAR-γ2基因Pro12Ala联合基因型分析结果：在本研究的NGT组和2DM组人群中共存在16种联合基因型形式。在汉族人群NGT组与T2DM组之间均无显著差异(P＞0.05)，其中较为常见的联合基因型(出现频率超过5％以上)有7种，依次为CC-GA-PA(25.7％与22.3％)、CA-GA-PP(20.0％与12.3％)、CA-GG-PP(15.7％与17.6％)、CC-GG-PP(12.9％与14.9％)、CC-AA-PP(8.6％与8.8％)、AA-GA-PP(5.7％与8.8％)AA-GG-PP(5.7％与5.4％)；在朝鲜族人群较为常见的联合基因型(出现频率超过5％以上)也有7种，依次为CC-GA-PA(29.1％与10.9％)、CC-GG-PP(19.8％与21.7％)、CA-GA-PP(17.4％与15.2％)、CA-GG-PP(8.1％与9.8％)、CC-AA-PP(8.6％与8.8％)、CA-AA-PP(7.0％与4.3％)、AA-GA-PP(4.7％与7.6％)，其中CA-GG-PP联合基因型频率NGT组较T2DM组显著增高(P=0.012)，表明此联合基因型可能是延边朝鲜族人群的保护型基因型，此基因型在汉族人群虽然NGT组较T2DM组有增高趋势，但无统计学意义(P=0.663)，在T2DM组的汉族较朝鲜族有增高趋势(基因型频率分别为22.3％和10.9％)，但也无统计学意义(P=0.058)，其他联合基因型在朝鲜族人群中均未发现与T2DM组存在显著性差异(P＞0.05)。3．利用传递不平衡(TDT)检测了九个T2DM核心家庭PPAR-γ2、αpM-1和PGC-1基因8个SNPs(-11377 C＞G、-11156 insCA、T45G、+276C＞A、+349A＞G及PGC-1的IVS2+52C＞A、Gly482Ser、Thr528Thr)结果：不支持此8个位点与延边朝鲜族和汉族2型糖尿病相连锁；初步筛查到由PGC-1基因IVS2+52C＞A、Gly482Ser和Thr528Thr多态构成的一个危险单倍型AAA，提示具有家族史的个体罹患2型糖尿病危险度提高5.6倍，但是，由于我们收集的核心家庭较少，还不能定论，有待于扩大收集的家系，继续深入研究。结论1．确定延边地区2型糖尿病主要危险因素是糖尿病家族史、腰臀比、高血压、甜食摄入量，支持2型糖尿病是多基因遗传模式。2．首次对中国延边朝鲜族和汉族PPAR-γ2、αpM-1和PGC-1三个T2DM候选基因进行部分基因片断扫描，检测到15个SNPs：Pro12Ala、C161T、-11391G＞A、-11377C＞G、-11156insCA、T45G(Gly15Gly)、+276C＞A、+349A＞G、R221S、H241P、Thr394Thr、IVS2+52C＞A、Gly482Ser、Thr528Thr、Ser577Leu；证实αpM-1基因的R221S、H241P、-11391G＞A和PGC-1基因的Ser577Leu为罕见基因；鉴定可能不属于本地区的5个SNPs：αpM-1基因+1711C＞T、+1795A＞G、+1833C＞A和PGC-1基因Ser74Leu和Asp475Asp。尚未发现的SNPs为PPAR-γ2基因的Pro115Gln、Gln438Gln、Ser457Ser、Leu481Leu、IVS2+40C＞T，αpM-1基因-11426G＞A、-11043C＞T、G 162A(Gly54Val)和PGC-1基因Ser74Leu、Leu410Iso、Leu438Ser。3．15个SNPs的基因型频率和等位基因频率在朝鲜族和汉族之间无差异。中国延边地区的朝鲜族和汉族基因频率不存在民族差异，遗传结构基本相同，亲缘性较近。但存在种族和地域差异。4．PGC-1基因IVS2+52C＞A和Gly482Ser以及PPAR-γ2基因C161T多态可能通过影响脂质代谢来触发2型糖尿病发生。可能是延边朝鲜族和汉族2型糖尿病危险因子。5．PPAR-γ2基因C161T、αpM-1基因-11377C＞G、-11156InsCA、T45G、+276C＞A、+349A＞G和PGC-1基因IVS2+52C＞A、Gly482Ser(G＞A)8个标记位点未与2型糖尿病连锁。更多还原

【Abstract】 OBJECTIVE Type 2 diabetes mellitus (T2DM) is a common polygenichereditary disease characterized as chronic hyperglycemia due to insulin resistance orsecretion decreases. It is also called complex disease. Hereditary factors play animportant role in the pathogenesis of T2DM. Both multiple minor genes andenvironment factors are responsible for the development of the disease. The purpose ofthis paper is to explore the risk factors and hereditary features of T2DM in Korean andHan ethnic nationality of Yanbian area in China, via the determination of the singlenucleotide polymorphisms (SNPs) of candidate genes of T2DM, the proxisomeproliferators activated receptor-γ, (PPAR-γ2), the adiopentin (αpM-1) and theperoxisome proliferator-activated receptor-γcoactivatorl (PGC-1), furthermore toanalyze their haplotypes and allele linkage disequilibrium.METHODS Part 1: A frequency matched case-control study was used base onthe epidemiological survey of T2DM in urban community population of Yanbian area inChina. 222 T2DM patients were taken randomly as case-group and 1747 as first-degreerelatives. Compared with case-group, 458 people having normal glucose tolerance wereselected as control-group and 2261 as first-degree relatives. The natural condition,living habit, patient history, symptoms, physical symptom, twenty items laboratoryexamination and genealogy atlas of all the persons tested were carried out. Falconer wasapplied to estimate heretability, and Li-Mantel-Gsrt to determine segregation ratio. Thehigh risk factors of T2DM were analyzed by unconditional univariate and multivariateLogistic regression. Part 2: The locations of genes of PPAR-γ2,αpM-1 and PGC-1were screened in Human SNPView of NCBI database and MapHap database using bioinformation methods. The 30 allele sites in this study were selected based on thehaplotype and haplotype tag SNP (htSNP) constructed in the database, as well asreported pappers. The genetic polymorphisms were determined by polymerase chainreaction-single strand conformational polymorphism (PCR-SSCP) and DNA sequencing.Case-contrast analysis was used to investigate the association between genepolymorphisms and gene frequencies in 269 T2DM patients (103 Korean and 156Chinese) and in 221 control subjects (108 Korean and 113 Chinese). The haplotype andlinkage disequilibrium of SNPs were analyzed using the SHEsis software on line(http://202.120.7.14/) in order to screen the risk factors of T2DM. Part 3: The SNPstransmission disequilibrium test (TDT) of 8 common sites of PPAR-γ2,αpM-1 andPGC-1 was conducted in the nuclear families of T2DM patients by using AssistantVersion 10.11 software (BioData, Ltd) in order to analyze the linkage between SNPsand T2DM.RESULTS Part 1: The prevalence rates of the T2DM first-degree relatives were5.09% and 4.82% respectively in Yanbian Korean and Han ethnic nationalities,significantly higher than those in control-group, which were 1.64% and 0.89% (P＜0.001). Prevalence relative risks (RR) were respectively raised 3.10 and 4.82 times.Among them, the first-degree relatives prevalence rate in Korean femalepatients(12.39%) was significantly higher than that in Korean male, Han ethnicnationality (4.67%, 4.85% and 4.80% respectively, P＜0.05). The first-degree relativesRR in Korean female patients were 2.6 times more than that in other relationships.Statistical results indicated the prevalence rate of T2DM was not significantly differentbetween Korean and Han ethnic nationality (P=0.809, 0.056). It illustrated nonationality difference in Yanbian, but having an evidently hereditary tendency. Totalprevalence rate of T2DM was 39.6% in Yanbian. Prevalence rate of Korean and Hanethnic nationalities were 39.4% and 51.6% respectively. Han ethnic nationality washigher than Korean nationality, but no significant difference. The first-degree relatives prevalence rate in Korean female (76.4%) was significantly higher than that in male(37.2%, P＜0.001). It pointed out that the first-degree relatives’ prevalence rate ofT2DM in Korean female had a hereditary liability than other people. The segregationratio of T2DM in Korean and Han ethnic nationality in Yanbian were 0.21 and 0.15,which were lower than 0.25, and not meet the features of single gene inheritance disease,and support the character of polygenic diseases. Family history of T2DM, hypertension,waist-to-hip ratio (WHR), intaking of sweet food were the major risk factors of T2DM.Part 2:15 gene polymorphisms were found from PPAR-γ2,αpM-1 and PGC-1 byPCR-SSCP and DNA sequencing. They were Pro12Ala and C161T (His477His) inPPAR-γ2; -11391 G＞A, -11377C＞G, -11156 insCA, T45G (Glyl5Gly), +276C＞A,+349A＞G, R221S and H241P inαpM-1; Thr394Thr, IVS2+52C＞A, Gly482Ser(G＞A),Thr528Thr(G＞A), Ser577Leu (C＞T) in PGC-1. Whereas, 15 SNPs were not detected inthis study. They were Pro115Gln, Gln438Gln, Ser457Ser, Leu481Leu, IVS2+40C＞T,-11426G＞A, -11043C＞T, +162G＞A(Gly54Val), +1711C＞T, +1795 A＞G, +1833 C＞A,Ser74Leu, Leu 410 Iso, Leu 438 Ser and Ser577Leu. The rare genes were -11391G＞A(A frequency 0.009), Ler475SerC＞T (T frequency 0.0013), R221SC＞A (A frequency0.003), H241P A＞C (C frequency 0.0015). The results meet the Hardy-Weinberg law.The genotype frequency and allele frequency of Pro12Ala in PPAR-γ2 in Han T2DMand NGT groups were PP: 0.954 and 0.960; PA: 0.046 and 0.040; A frequency 0.023and 0.020. However, those in Korean T2DM and NGT groups were PP: 0.948 and 0.955;PA: 0.052 and 0.045; A frequency 0.026 and 0.022. No difference was found betweenT2DM and NGT groups. The genotype frequency and allele frequency of 5 SNPs inαpM-1 in T2DM and NGT groups were G in -11377C＞G: 0.237 and 0.225; I in-11156insCA: 0.132 and 0.158; G in T45G: 0.294 and 0.389; T in +276G＞T: 0.294 and0.289; G in +349A＞G: 0.324 and 0.389. There was no difference. We also failed to findthe difference in the genes IVS2+52C＞A, Thr394Thr, Gly482Ser(G＞A) and Thr528Thr(G＞A) in PGC-1 between Han and Korean. The combined genotypes CC-GA-PA, CA-GA-PP, CA-GG-PP, CC-GG-PP and CC-AA-PP in IVS2+52C＞A and Gly482Ser ofPGC-1 as well as in Prol2Ala of PPAR-γ2 appeared in high frequency (over 5%).Part 3: The 8 SNPs(-11377C＞G, -11156 insCA, Gly15Gly (T45G), +276C＞A,+349A＞G, IVS2+52 C＞A, Gly482Ser and Thr528Thr from PPAR-γ2, apM-1 andPGC-1 were not associated with T2DM in Han and Korean as showed in TDT.CONCLUSION (1) The major risk factors of T2DM in Yanbian area are family history,hypertension, WHR and intaking of sweet food. The hereditary character of T2DM ispolygenic. (2) 15 SNPs: Prol2Ala, C161T, -11391G＞A, -11377C＞G, -11156insCA, T45G(Gly15Gly), +276C＞A, +349A＞G, R221S, H241P, Thr394Thr, IVS2+52C＞A,Gly482Ser(G＞A), Thr528Thr(G＞A) and Ser577Leu(C＞T) in PPAR-γ2, apM-1 andPGC-1 related with T2DM were detected in Han and Korean in Yanbian of China. (3)There were no differences in genotype frequency and allele frequency of 15 SNPsabove between Han and Korean nationalities in Yanbian of China. (4) Thepolymorphisms of IVS2+52C＞A and Gly482Ser in PGC-1, C161T in PPAR-γ2 wereassociated with T2DM, possibly by interfering with lipid metabolism. (5) Thegenotypes C161T, -11377C＞G, -11156InsCA, T45G, +276C＞A, +349A＞G. IVS2+52C＞A and Gly482Ser(G＞A) in PPAR-γ2, apM-1 and PGC-1 were not associated withT2DM in Yanbian Han and Korean patients.更多还原