【作者】 柴莉；

【导师】 霍正浩；

【作者基本信息】 宁夏医科大学 ， 遗传学， 2010， 硕士

【摘要】 目的本研究对细胞毒性T淋巴细胞相关性抗原-4 ( cytotoxic T lymphocyte-associated antigen-4, CTLA-4)基因SNP-1722(T/C)、SNP-1661(A/G)、SNP-318(C/T)和SNP+49(A/G)四个多态性位点进行分析,探讨CTLA-4基因四个多态性位点与宁夏汉族反复自然流产(RSA)的相关性,研究CTLA-4基因四个SNP位点构建的单倍型与反复自然流产的关联性。方法选取2006年1月-2009年7月在宁夏医科大学生殖与遗传实验室进行染色体检查的反复自然流产者242例做为病例组,年龄19-42岁,平均年龄为27.97±4.49岁;选取2008年1月-2009年8月在银川市妇幼保健院住院分娩的正常孕妇228例做为对照组,年龄20-36岁,平均年龄为27.67±3.67岁。用聚合酶链反应-限制性片段长度多态性(polymerase chain reaction- restricted fragment length polymorphism, PCR-RFLP)技术检测病例组与对照组CTLA-4基因四个位点多态性。SPSS11.5软件计算基因型频率,等位基因频率及比值比(OR),比较各组基因型频率和等位基因频率及进行相对危险度的估计,分析CTLA-4基因四个SNPs位点与反复自然流产的相关性,SHEsis软件构建单倍型探讨CTLA-4基因四个SNPs位点与反复自然流产的易感性是否有关联。结果①经遗传平衡定律检验,研究对象CTLA-4各基因型频率分布均符合Hardy-Weinberg平衡法则,具有群体代表性。②CTLA-4基因SNP-1722、SNP-1661、SNP+49位点各基因型频率和等位基因频率在病例组与对照组中的分布无统计学意义(P>0.05);CTLA-4基因SNP-318(C/T)的CC基因型在病例组中的分布频率明显高于对照组,CT基因型在病例组中的分布频率明显低于对照组,差异具有统计学意义(P=0.003,OR=1.941;P=0.007,OR=0.546),而TT基因型在病例组和对照组之间差异无统计学意义(P=0.345,OR=0.237);卡方检验后,C等位基因在病例组中的频率明显高于对照组,而T等位基因在病例组中的频率明显低于对照组,差异均具有统计学意义(P=0.002,OR=1.853;P=0.002,OR=0.540)。③CTLA-4基因四个SNPs位点之间两两连锁不平衡(D’>0.8)。④CTLA-4基因SNP-1722、SNP-1661、SNP-318和SNP+49四个位点构建的单倍型中有9种保护性单倍型在病例组与对照组中具有统计学意义(P<0.05,OR<1);2种危险致病单倍型在两组中的分布具有统计学差异(P<0.05,OR>1)。结论1、CTLA-4基因SNP-1722、SNP-1661、SNP+49多态性位点各基因型频率和等位基因频率在病例组与对照组中的分布无统计学意义,可能与宁夏汉族女性反复自然流产的易感性无关。2、CTLA-4基因SNP-318多态性与反复自然流产可能有一定的关联性,其CC、CT基因型可能是宁夏汉族女性反复自然流产的易感因素,同时携带T等位基因的个体发生反复自然流产的风险明显降低。3、CTLA-4基因四个SNPs位点两两之间连锁不平衡。4、CTLA-4基因构建的单倍型中,有9种单倍型很可能是宁夏汉族女性反复自然流产的保护性因素,2种单倍型可能是宁夏汉族女性反复自然流产的危险性因素。目的研究细胞毒性T淋巴细胞相关抗原-4(CTLA-4)基因启动子区SNP-1722(T/C)、SNP-1661(A/G)、SNP-318(C/T)和第一外显子SNP+49(A/G)四个多态性位点在宁夏回族-汉族群体中的基因型频率和等位基因频率分布特征。方法来源于宁夏医科大学附属医院健康体检者,回族208人(回族女性119人,回族男性89人),汉族317人(汉族女性223人,汉族男性94人),平均年龄(24.42±3.69)岁。均无慢性病史、无放射和化学药物治疗史、无自身免疫性疾病史,祖籍为宁夏的回族和汉族群体。应用PCR–RFLP技术对宁夏回族和汉族群体CTLA-4基因启动子区SNP-1722(T/C)、SNP-1661(A/G)、SNP-318(C/T)位点及第一外显子区SNP+49(A/G)位点分布进行检测。SPSS11.5软件计算基因型频率及等位基因频率,比较回-汉群体基因型频率和等位基因频率的差异,分析CTLA-4基因四个SNPs位点在宁夏回族-汉族群体中分布特征。结果宁夏回族与汉族群体的CTLA-4基因SNP-1722、SNP-1661、SNP-318、SNP+49位点基因型频率及等位基因频率比较差异均无统计学意义(P>0.05)。合并宁夏回汉族群体,按性别重新分组后比较,显示CTLA-4基因SNP-1661,SNP-318,SNP+49三个位点基因型频率及等位基因频率差异均无统计学意义(P>0.05),而CTLA-4基因SNP-1722位点TT、TC和CC基因型频率在性别组间差异有统计学意义(χ2=9.280,P=0.010),其中CC基因型频率女性明显高于男性(16.08%vs7.11%,χ2=8.523,P=0.004),C等位基因频率从女性与男性的比较的P值看差异无统计学意义(χ2=2.162,P=0.141),但是显示C等位基因含量具有女性较男性高。在国内不同地区之间CTLA-4基因SNP-1722、SNP-1661、SNP-318、SNP+49位点基因型频率及等位基因频率分布特征不尽一致;与不同国家和人种之间比较也得出相似结果。结论宁夏回族与汉族群体的CTLA-4基因SNP-1722、SNP-1661、SNP-318、SNP+49位点基因型频率及等位基因频率分布特征可能相同,但SNP-1722位点CC基因型在男女之间存在差异。同时CTLA-4基因SNP-1722、SNP-1661、SNP-318、SNP+49多态性位点在国内不同地区,不同国家存在地域和种族差异。更多还原

【Abstract】 Objective To investigate the correlation between 4 SNPs (SNP-1722(T/C)、SNP-1661(A/G)、SNP-318(C/T) polymorphisms in promotor region and the SNP+49(A/G) polymorphisms in exon 1 region of the cytotoxic T lymphocyte-associated antigen-4 gene) and recurrent spontaneous abortion (RSA) in Chinese women. By compare genotype distribution and allele frequency between the patient of recurrent spontaneous abortion (RSA) and normal pregnancy women, to approach the relationship about the haplotype of four SNPs of the cytotoxic T lymphocyte-associated antigen-4 gene with RSA.Methods 242 patients of RSA were recruited from NingXia medical university genetic laboratory as study group with an average age of 27.97±4.49. 228 normal pregnancy women from Maternal and Child Health Hospital were as control group with an average age of 27.67±3.67. The Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) were used to analyse the genotype distribution and allele frequency of promoter region -1722, -1661, -318 and exon 1 region +49 positions in CTLA- 4 gene. The statistical analysis included chi-square test and SPSS11.5 software was used to analyze the data and calculated the distribution of genotype and frequency of allele. Hardy-Weinberg balance, linkage disequilibrium and haplotype analyzed by using software SHEsis.Results①The genotype distribution of the 4 SNPs in CTLA-4 gene reached Hardy-Weinberg equilibrium, indicated that the population in present study were selected randomly.②There were no significantly different in genotype distribution and allele frequencies of CTLA-4 gene SNP-1722, SNP-1661, SNP+49 between the patient of RSA and normal pregnancy women (P>0.05).However, in case group, the frequency of CC genotype at the -318 position was statistically higher than controls, and the frequency of CT genotype was lower than controls (P=0.003,OR=1.941;P=0.007,OR=0.546); and the frequency of C allele in case was higher than control, but the frequency of T allele was lower than control (P=0.002,OR=1.853;P=0.002,OR=0.540).③Each pair of four SNPs in CTLA-4 gene were linkage disequilibrium (D’>0.8).④Among the haplotypes established by the 4 SNPs, there were nine kinds of protective haplotypes significantly different between case and control (P<0.05, OR<1), and two kinds of rise haplotypes also have significant difference between case and control (P<0.05, OR>1).Conclusions (1) There were no significant differences in genotype distribution and allele frequencies of CTLA-4 gene SNP-1722, SNP-1661, SNP+49 between two groups. These polymorphisms locus may not associated with the susceptibility of RSA in NingXia female. (2) CTLA-4 gene SNP-318 polymorphism may have some relevance with RSA, and the CC, CT genotype probably associated with the susceptibility of RSA in NingXia female. Individuals who carry T allele the risk of occur RSA will be significantly decreased. (3) Each pair of 4 SNPs were linkage disequilibrium. (4) The four SNPs in CTLA-4 gene may have nine kinds of protective haplotypes and two kinds of rise haplotypes. Objective To study the distribution traits of genotype and allele frequencies of cytotoxic T lymphocyte-associated antigen-4 gene promoter region SNP-1722(T/C), SNP-1661(A/G), SNP-318(C/T) and exon 1 region SNP+49(A/G) polymorphism in Ningxia Hui-Han population.Methods 525 healthy people from NingXia medical university affiliated hospital were slected as study group with an average age of 24.42±3.69. (208 hui population and 318 Han population). All subjects have no history of chronic disease, radio-chemotherapy and autoimmunity disease, ancestry are hui and han nationality. PCR-RFLP method was used to determine the distribution of CTLA-4 gene promoter region SNP-1722(T/C), SNP-1661(A/G), SNP-318(C/T) and exon 1 region SNP+49(A/G) polymorphisms in Hui-Han population. The distribution of genotype and frequency of allele were calculated by SPSS11.5 software to analyze the difference of CTLA-4 gene four SNPs sites between hui and han population. Hardy-Weinberg of the four SNPs sites were evaluated by SHEsis software.Results There were no significantly different in genotype frequencies and allele frequencies of CTLA-4 gene SNP+49, SNP-318, SNP-1661, SNP-1722 between Hui and Han population (P>0.05). With the combination of Hui and Han population of Ningxia, and then divide into two groups as sex, the genotype distribution and allele frequency of CTLA-4 gene SNP-1661, SNP-318, SNP+49 have no significant difference between Hui and Han population (P>0.05), but the genotype frequencies at -1722 position have significant difference between female and male (χ2=9.280,P=0.010) , and the CC genotype in female was higher than male (16.08%vs7.11%,χ2=8.523,P=0.004). Although there was no significant difference between female and male in the frequency of the C allele at -1722 position (χ2=2.162, P=0.141), it shows that female tend to carry more C allele. The distribution traits of genotype and allele frequencies of cytotoxic T lymphocyte-associated antigen-4 gene of SNP-1722, SNP-1661, SNP-318, SNP+49 polymorphism sites were not all consistent in different Chinese area, as well as in other different contries and races.Conclusions The distribution traits of allele and genotype frequencies of CTLA-4 gene SNP+49, SNP-318, SNP-1661, SNP-1722 positions may be similar with Hui and Han population of Ningxia, but there was significant difference between female and male in the frequency of the CC genotype at -1722 position. Meanwhile, the CTLA-4 gene SNP-1722, SNP-1661, SNP-318, SNP+49 polymorphisms sites have significantly different among different area and races in China and different contries.更多还原