【作者】 江波；

【导师】 张日；

【作者基本信息】 苏州大学 ， 内科学， 2010， 博士

【摘要】 【背景与目的】供者杀伤细胞免疫球蛋白样受体(KIR)基因簇和受者人白细胞抗原(HLA)不匹配诱导的NK细胞异体反应可改善异基因造血干细胞移植的治疗效果。供者KIR和受者HLA I类分子发生错配时,KIR抑制基因KIR3DL1、KIR2DL1和KIR2DL2/3不能阻断NK细胞的细胞毒作用,而表现出异体反应直接杀伤受者的白血病细胞、T细胞和树突状细胞,进而降低白血病复发率、增加移植物存活率、降低急性移植物抗宿主病的发生率和严重程度。2002年Ruggeri等人在《Science》杂志报道后,陆续有多个血液中心进行类似的报道。KIR3DL1、KIR2DL1和KIR2DL2/3在基因水平、等位基因水平和受配体系统都具有极其复杂的多态性。目前国内实验室仅能在基因水平进行研究,尚不能在等位基因水平开展工作。国际上也仅少数几个实验室能够开展KIR基因DNA编码序列的分析研究工作,但是其方法仍不十分完善。为此我们在中国苏州大学附属第一医院和美国乔治城大学医学部探索KIR3DL1、KIR2DL1和KIR2DL2/3可靠的高分辨检测方法,并从基因水平、等位基因水平和受配体系统三个层面进行系统研究。【方法】第一部分90个蒙古族健康人群全血标本采集自内蒙古通辽市科尔沁草原牧区,提取DNA,以两组不同引物对KIR基因簇进行序列特异性引物聚合酶链反应(PCR-SSP),同时设置阳性对照、阴性对照和空白对照,以GH或β-actin为内参。扩增产物进行琼脂糖凝胶电泳。对少数两套引物扩增结果不一致的样本使用第三套引物进行确认。根据扩增的结果计算人群中KIR基因簇各基因的携带率、查询各个体的基因型、计算观察基因遗传的连锁不平衡现象。收集其他24个人群的相关数据,统计分析绘制主成分分析散点图和系统进化树。第二部分100名非裔美国人样本B细胞经EB病毒转染构建细胞株后,常规培养、提取DNA。设计特异性引物、摸索扩增条件对KIR3DL1和KIR2DL1进行大片段的扩增和测序。KIR2DL2/3DNA样本扩增前需先使用单倍型磁珠分离技术和/或限制性内切酶进行预处理,去除干扰测序结果的相似基因,再使用特异性引物进行大片段扩增和测序。第三部分高分辨测定非裔美国人HLA-A、HLA-B和HLA-C的等位基因结果,根据获得的NMDP编码查询各等位基因是否携带HLA-Bw4抗原决定簇和HLA-C1、C2的分组。对照KIR3DL1、KIR2DL1和KIR2DL2/3的实验结果分析受配体系统的多态性。第四部分根据KIR3DL1、KIR2DL1和KIR2DL2/3的高分辨结果,对疑似新等位基因的样本进行分离。使用等位基因特异性引物扩增、单倍型磁珠分离和PCR分子克隆技术对等位基因进行分离、扩增和测序,将获得的新等位基因序列向Genebank和WHO申报,申请新等位基因的命名。【结果】1、所有KIR基因均可在蒙古族人群中发现,其中框架基因KIR3DL2、KIR3DL3携带率为100%。一个样本缺失KIR2DL4基因。KIR2DL1、KIR2DL3、KIR3DL1携带率分别为93.3%、93.3%和95.6%,KIR2DL2的携带率较低为30.0%。KIR2DL2的携带率介于蒙古利亚人和高加索人之间。蒙古族人群中37.78%个体的基因型为AA,介于蒙古利亚人和高加索人之间。KIR2DL2和KIR2DS2存在连锁遗传关系,KIR2DL5、KIR2DS1、KIR2DS3和KIR2DS5之间也存在有连锁遗传关系。2、主成分分析作蒙古族人群和其他24个人群的散点图见高加索人包括欧洲人、欧裔美国人、阿根廷人、黎巴嫩人及中国的维吾尔族人在散点图中部聚集,蒙古利亚中的上海汉族、云南汉族、云南彝族、日本人、韩国人及新加坡汉族在散点图左侧聚集,而蒙古族人群则位于两大人种之间。Physical6.22系统进化软件包构建系统进化树也发现有类似现象。3、建立使用相互重叠、可以相互验证的大片段扩增测序方法,获得较满意的结果。KIR3DL1、KIR2DL1和KIR2DL2/3的测序结果和欧裔美国人的相关研究结果进行对照研究。非裔美国人中携带KIR3DL1的比例为99%,KIR3DL1*01501和KIR3DL1*01502是最常见等位基因。KIR3DL1*002, *00402, *008, *019,和*029存在于非裔美国人群中,说明了非裔美国人祖先移民美国后,经过数百年历史已逐步混入了其他种族的KIR3DL1等位基因。发现有3个样本携带有KIR3DL1*059,KIR3DL1*059为KIR3DL1和KIR3DL2的融合基因(KIR3DL1/2v),有2个样本为KIR3DL1/2v的新等位基因需进行分离和分析。4、3%的非裔美国人未携带有KIR2DL1基因。42个样本携带有1个KIR2DL1基因或2个相同的等位基因,55个样本是不同等位基因的杂合子。KIR2DL1*00302和KIR2DL1*00401是非洲人群中最常见的等位基因,携带率分别是68%和22%,KIR2DL1*006, KIR2DL1*007在非裔美国人中携带率为11%和7%。5、非裔美国人中KIR2DL2*001携带频率较多,而KIR2DL2*003的携带率较低,KIR2DL2*00602为非洲人较特异的等位基因。KIR2DL3中KIR2DL3*001在非裔美国人和欧裔美国人中均占较高比例>60%,但是在非洲人中KIR2DL3*002中携带较低。6、19个样品中含有KIR3DL1基因但其HLA系统中不含有可和KIR3DL1结合的Bw4抗原决定簇。1个样本含有纯合的KIR3DL1*004,均不能诱导生成具有细胞毒作用的KIR3DL1+NK细胞。25%的样品中不含有HLA-C2组的等位基因,有32%的样品中不含有HLA-C1组的等位基因。43%的人既含有HLA-C1又含有HLA-C2组的等位基因。7、7个KIR3DL1新等位基因及11个KIR2DL1新等位基因被使用不同方法成功分离,测序获得全长DNA编码序列。【结论】1、从基因水平分析蒙古族人群的KIR基因簇多态性。蒙古族人群活化基因携带率高于蒙古利亚人低于高加索人;蒙古族人群haplotype AA基因型携带率低于蒙古利亚人,高于高加索人;统计学主成分分析和无根系统进化树证明见蒙古族人群介于高加索人和蒙古利亚两大人种之间。蒙古族人群似同时具备了高加索人和蒙古利亚的遗传特征。2、从等位基因水平分析研究KIR基因簇抑制基因在非裔美国人中的多态性。非裔美国人的KIR3DL1抑制基因等位基因水平的分布特征说明了非裔美国人和非洲人群有较近的亲缘关系。3、非裔美国人中有3个(3%)未携带有KIR2DL1基因。KIR2DL1*00302和KIR2DL1*00401是非洲人群中最常见的等位基因。4、非裔美国人中KIR2DL2*001携带频率较高,而KIR2DL2*003的携带率较低,KIR2DL2*00602为非裔美国人较特异的等位基因。KIR2DL3中KIR2DL3*001在非裔美国人中最为常见,KIR2DL3*002中携带较低。5、从KIR-HLA受配体水平分析和研究KIR基因簇受配体系统的多态性,发现了在非裔美国人中19%缺失HLA-Bw4抗原决定簇。25%的非裔美国人为两个HLA-Cw等位基因均为HLA-C1,32%的人HLA-Cw等位基因均为HLA-C2,只有43%的人既含有属于HLA-C1组的等位基因又含有属于HLA-C2组的等位基因。6、本研究成功发现和分离了7个KIR3DL1新等位基因和11个KIR2DL1的新等位基因,已成功申报Genebank,并获得WHO的正式命名。更多还原

【Abstract】 Background and ObjectiveDonor killer cell immunoglobulin-like receptors (KIR) cluster and recipient human leukocyte antigen (HLA) mismatching induce NK cell alloreactivity in allo-hematopoietic stem cell transplantation. NK cells alloreactivity kill recipient leukemia cells, T cells and dendritic cells, also improve HSCT outcome by reducing the relapse rate, increasing graft survival, reducing acute graft-versus-host disease incidence and severity. Donor selection by testing KIR gene cluster can effectively improve HSCT outcome. KIR3DL1, KIR2DL1 and KIR2DL2/3 mismatching improved HSCT outcome has been confirmed. However, domestic labs only are capable of typing KIR genotype, can’t typing KIR cluster in allele level. There are few laboratories can investigate KIR gene DNA full-length sequence.So we designed this research to develop KIR3DL1, KIR2DL1 and KIR2DL2/3 high resolution typing methods, systematically study these genes in the genotype level, allele level and ligand-receptor system in China and Georgetown University, United States.MethodsPart I 90 whole blood samples of healthy Mongolian donors were collected from Horqin prairie Tongliao City, Inner Mongolian. Genomic DNA was extracted following manufacture protocol. Each sample was amplified by two published KIR gene sequence specific primer sets, while amplified positive control, negative control and blank control,GH orβ-actin was amplified as the internal control. PCR products were checked by agarose gel electrophoresis. A few samples have discrepancy results by primer set A and set B. The third set of primers was used to confirm amplified results. KIR gene frequency and genetic linkage disequilibrium have been calculated according to the results of amplification. Individual genotype was searched from a website. Mongolian KIR frequency and 24 published population data were analyzed by SAS8.0. Scatter gram was constructed by Principal component analysis. Genetics tree was drew according to Nei genetic distance by Phylogenetic Software.Part II 100 African-American samples’B cell lines were transfected by EB virus. Genetic DNA was extracted. KIR3DL1 and KIR2DL1 were amplified by 2~3 overlapped pieces to cover whole DNA sequence. KIR2DL2/3 should be prepared by haploprep beads and/or restriction enzyme digestion to remove similar gene before amplified. Each amplicon was typing by bigdye and sequence primers.Part III HLA-A、HLA-B or HLA-C was amplified from genomic DNA. After purified by magnetic beads, amplicons were sequence by sequence reaction. According to available NMDP allele code, Samples were checked in the website to acquaint whether it’s HLA-A、B loci carried HLA-Bw4 epitope and HLA-C classificationPart IV KIR3DL1, KIR2DL1 and KIR2DL2/3 high-resolution results showed some samples had suspicious new alleles. Each new allele should be separated to confirm. Alleles were separated by allele-specific primer PCR, haploprep beads and PCR product cloning, then amplified and sequenced as same as allele typing. New alleles were calculated amino code alteration and submitted to Genebank and WHO.Results1 All KIR genes were found in Mongolian population. The framework genes KIR3DL2, KIR3DL3 frequency was 100%. A sample lack KIR2DL4 gene. The frequencies of KIR2DL1, KIR2DL3 and KIR3DL1 were 93.3%, 93.3% and 95.6%, respectively. The frequency of KIR2DL2 was lower as 30.0%, between Mongoloid and Caucasoid. The frequency of haplotype AA in Mongolian population was 37.78%, also between Mongoloid and the Caucasoid. KIR2DL2-KIR2DS2 and KIR2DL5-KIR2DS1-KIR2DS3-KIR2DS5 showed linkage disequilibrium.2 Principal component analysis for Mongolian populations and other 24 populations, constructed scatter diagram. Caucasoid populations: Europeans, European Americans, Argentinean, Lebanese and Chinese Uighurs gathered in the central, Mongoloid populations: Chinese Han in Shanghai, Yunnan Han, Yunnan Yi, Japanese, Korean and Singapore Han in the left of the diagram, while the Mongolian population was located between Caucasoid and Mongoloid. Genetic tree constructed by Physical6.22 Package also showed a similar result. 3 KIR3DL1, KIR2DL1 and KIR2DL2/3 were amplified by large, overlapped pieces and sequenced and compared to European Americans typing results. The frequency of KIR in African-American population was 99%. KIR3DL1*01501 and KIR3DL1*01502 is the most common alleles in African-American population. KIR3DL1*002, *00402, *008, *019 and *029 existed in the African-American population, but wasn’t found in African population proved that African-American has gradually mixed with other races KIR3DL1 alleles after their ancestors emigrated to American. Three samples carrying heterozygous KIR3DL1*059, an allele derived from KIR3DL1 and KIR3DL2 fusion gene. There were two alleles similar to KIR3DL1*059 should be separated and analyzed.4 3% African American population didn’t carry any KIR2DL1 allele. 42 samples carried an allele or homozygous alleles. 55 samples carried heterozygous KIR2DL1 alleles. KIR2DL1*00302 and KIR2DL1*00401 were the most popular alleles in African population, their frequencies were 68% and 22%.The frequency of KIR2DL1*006, KIR2DL1*007 in African-Americans were 11% and 7%.5 African Americans carried more KIR2DL2*001 than European Americans, and KIR2DL2*003 carried less. KIR2DL2*00602 was specific allele of African Americans. KIR2DL3*001 in African Americans and in European Americans were counted over 60%, but African American carried less KIR2DL3*002.6 19 samples contained KIR3DL1 genes, but not be combined with HLA Bw4 epitope. A sample contained homozygous KIR3DL1*004, which can’t expressed in the surface of NK cells, and can’t induced KIR3DL1+ functional NK cells. 25% of the samples did not contain HLA-C2 group alleles, with 32% of the samples did not contain HLA-C1 group alleles. 43% of the people have both HLA-C1 group and HLA-C2 group alleles.7 7 KIR3DL1 novel alleles and 11 KIR2DL1 novel alleles were successfully isolated by different methods. The whole DNA sequences were typing and submitted to WHO.Conclusions1 To Study diversity of KIR cluster in Mongolian population on the genotype level. Mongolian population carries more activating KIR genes than Mongoloid, but less than Caucasoid. The frequency of haplotype AA in Mongolian population was lower than Mongoloid, higher than Caucasoid. Principal component analysis and genetic tree without roots showed Mongolian population between Caucasoid and Mongoloid. Mongolian population had both genetic characteristics from Caucasoid or Mongoloid.2 To study polymorphism of KIR inhibitory genes in African Americans on the allele level. The allele distribution of inhibitory gene KIR3DL1 showed that African-Americans and African people have a closer relationship.3 3% donors in African Americans did not carry KIR2DL1. KIR2DL1*00302 and KIR2DL1*00401 is the most common allele in African American population Hou L, Chen M, Jiang B4 The frequency of KIR2DL2*001 was the highest in African Americans. KIR2DL2*00602 is a specific allele in African Americans. KIR2DL3 * 001 is the most common in African-Americans.5 Analyzing KIR inhibitory genes and their HLA ligands was found that 19% of African-Americans lack HLA Bw4 epitope. 25% of African Americans have homozygous HLA-C1 group alleles, 32% have homozygous HLA-C2 group alleles. Only 43% people carried heterozygous HLA-C1 group and HLA-C2 group alleles.6 7 KIR3DL1 novel alleles and 11 KIR2DL1 alleles were successfully isolated and submitted to Genebank and WHO. WHO have given official names to these new alleles.更多还原