肿瘤转移抑制基因HTPAP单体型与肝癌转移潜能的关系
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摘要
肝细胞癌(Hepatocellular carcinoma,以下简称肝癌,HCC)是世界上最常见、恶性程度最高的肿瘤之一,其病死率在全球所有癌症中占第3位,是我国第2位癌症死亡原因。手术切除仍是目前肝癌获得长期生存的主要手段。但术后5年复发率高达60%以上(小肝癌也达40%～50%)。转移复发己成为进一步延长肝癌病人生存期、提高远期疗效的瓶颈问题,也是最终攻克肝癌的关键。因此,筛选肝癌转移相关基因,探明肝癌转移的分子机制,寻找有效的防治手段成为进一步提高肝癌治疗效果的关键。
HTPAP基因是肿瘤相关基因,其正式名为PPAPDC1B(Phosphatidic acidphosphatase type 2 domain containing 1B),含有1B功能域的2型磷脂酸磷酸酯酶。该基因定位于8p12,含有7个外显子、6个内含子。基因全长4459 bp,cDNA长为826 bp,编码一个具有175个氨基酸的蛋白质。已有研究发现一些人类肿瘤中该基因的异常转录表达,提示该基因具有抑制肿瘤细胞增殖、降低侵袭性、抑制肿瘤转移的作用。我们前期经体内、体外研究初步证实该基因可抑制HCC的侵袭和转移,提示HTPAP基因是一新的HCC转移抑制基因。
前期研究中,我们初步通过对肝癌病人肿瘤组织HTPAP基因的单核苷酸多态性分析研究发现,HCC病人HTPAP基因上的rs1149位点存在C→G多态性。伴有肝内播散和/或脉管侵犯的病人血浆循环DNA rs1149位点GG+GC基因型频率和G等位基因频率分别为61%和39%,明显高于不伴有肝内播散和脉管侵犯的病人该位点GG+GC基因型频率和G等位基因频率(分别为39%和24%),二组差异显著(P=0.046,P=0.026)。rs1149位点GG+GC基因型与肿瘤数目P=0.026),TNM分期(P=0.026),病人无瘤生存率(P=0.005)和总体生存率(P=0.014)均密切相关。rs1149位点上的C→G多态性可能影响HTPAP基因的表达或引起基因编码的蛋白质及其功能的改变,从而导致HCC病人肿瘤侵袭性的增高。
基于上述理论和实验基础,本实验拟应用激光捕获组织显微切割获取纯肝癌组织DNA,对HTPAP基因的SNP位点进行检测并行单体型构建,探讨HTPAP基因的SNP、单体型与肝癌转移潜能的关系及其相关机制,以及与肝癌病人转移复发和预后的关系。
第一部分肿瘤抑制基因HTPAP的SNP检测、单体型构建及其与肝癌侵袭性能的关系探讨
目的:在前期研究基础上通过对大样本的HCC病人肿瘤组织DNA的HTPAP基因的单核苷酸多态性(single nucleotide polymorphisms,SNPs)检测、单体型构建后,探讨其与肝癌侵袭性关系。
方法:应用激光捕获组织显微切割从864例HCC手术样本中获取相对纯的HCC细胞、提取DNA,对30例HCC的HTPAP基因全长和5'、3'-flanking region(侧翼区)的3kb启动子区域进行ABI仪测序发现SNP位点后,应用焦磷酸测序仪行864例样本SNP检测,并予单体型构建,分析其与肝癌侵袭转移潜能(有无肝内播散,脉管癌栓)的关系。
结果:在7.5 Kb测序区域中,共发现6个SNP位点[-1053 A/G、+64G/C、+357C/G、+1648-/TAAG(无插入和插入TAAG等位基因分别以T/G示,后同)、+1838A/G、+3528C/T]。单体型构建研究发现HTPAP中共存在12种单体型,其中A-G-C-T-A-C,G-C-G-G-G-T,A-G-C-T-G-C,G-C-G-G-A-T(依次为6个SNP的等位基因:+3528C/T—+1838A/G—+1648-/TAAG—+357C/G—+64G/C—-1053A/G,后同)四种单体型占94.62%。793例(占91.78%)HCC由该4种单体型构成。5个SNP(-1053 A/G、+64G/C、+357C/G、+1648-/TAAG、+3528C/T)表现为强连锁不平衡关系(r~2=0.85-1.0),而+1838A/G出现重组现象,与上述5个SNP连锁较弱(r~2=0.32-0.36);临床病理特征分析提示SNP+357C/G的基因型GG+GC与HCC高侵袭转移潜能正相关[GC:OR=1.77,95%C.I.=1.32-2.36,P=0.0001;GG:OR=2.11(1.28-3.48),P=0.003],呈等位基因G剂量依存性。而+1838A/G虽然处于外显子之中,但是与HCC侵袭转移潜能并无相关[GA:OR=1.09(0.81-1.46),P=0.564;GG:OR=1.39(0.91-2.12),P=0.126]。单体型G-C-G-G-G-T与高侵袭转移潜能正相关[OR=1.50(1.18-1.91),P=0.001],而G-C-G-G-A-T,A-G-C-T-A-C和A-G-C-T-G-C则与HCC侵袭性无关。
结论:肿瘤转移抑制基因HTPAP的TagSNP+357 GG+GC基因型和G-C-G-G-G-T单体型与HCC高侵袭转移潜能正相关,针对不同SNP基因型和单体型分析可能会为肝癌转移复发、预后预测提供新的途径和分子遗传学指标。
第二部分HTPAP基因的SNP基因型和单体型影响肝癌侵袭转移潜能的相关机制研究及其对预后的影响
一、HCC中HTPAP单体型与基因表达关系
目的:检测HCC中HTPAP基因的表达,探讨HTPAP基因的TagSNP+357C/G基因型和相关的单体型对与基因表达的的影响。
方法:(1)构建pEGFP-HTPAP载体真核表达质粒,应用绝对定量PCR(quantitative real-time PCR)检测HCC中HTPAP RNA的表达(含3种同素异构体).(2)随机选取170例以A-G-C-T-A-C,G-C-G-G-G-T,A-G-C-T-G-C和G-C-G-G-A-T等四种HTPAP基因主要单体型组成的HCC组织,并提取总RNA。
结果:成功构建pEGFP-HTPAP载体真核表达质粒。对4种单体型组成的170例HCC进行HTPAP基因表达水平的定量分析,其中GCGGGT纯合子21例、AGCTAC纯合子65例、AGCTAC/GCGGGT杂合子52例、AGCTAC/GCGGAT杂合子13例、AGCTAC/AGCTGC杂合子19例。发现HCC中HTPAP基因表达在AGCTAC纯合子和AGCTAC/AGCTGC杂合子病人较GCGGGT纯合子、AGCTAC/GCGGGT杂合子和AGCTAC/GCGGAT杂合子表达升高,但未达到统计意义(P＞0.05)。
结论:HCC中不同HTPAP单体型能影响HTPAP表达水平,但是无统计学差异。
二、HCC中HTPAP基因启动子单体型与转录活性的关系
目的:通过构建HTPAP的3个启动子单体型,探讨不同HTPAP启动子单体型对基因转录活性的影响。
方法:PCR方法克隆出跨越基因上游5'-flanking的-1764bp至基因内部+315bp的启动子序列(2079bp)。该区域含-1053AG和+64GC两个SNP,由于存在完全LD,故只有三种单体型(-1053 AA/+64GG、-1053 AG/+64 GC和-1053 GG/+64 CC),上述HTPAP启动子单体型pGL3质粒,并分别转染MHCC-97H、HepG2肝癌细胞和Hela细胞,采用双荧光报告法,检测荧光素酶的活性,重复3次。
结果:三种单体型的HTPAP启动子都具有显著转录活性(与空pGL3-basic质粒相比),其中-1053 AA/+64GG单体型启动子转录活性明显强于-1053 GG/+64 CC(P=0.005)和-1053 AG/+64 GC单体型启动子(P=0.009)。而-1053GG/+64 CC和-1053 AG/+64 GC启动子单体型间未见明显差异(P=0.26)。
结论:HTPAP启动子SNP(-1053AG和+64GC)的不同单体型对基因的转录活性有显著影响,能启动不同水平的基因转录。
三、利用基因芯片研究HTPAP单体型调控HCC侵袭转移潜能的调控机制
目的:应用表达谱芯片分析不同单体型HCC的差异表达,探不同单体型HTPAP基因影响HCC的侵袭性潜能可能机制。
方法:选择GCGGGT和AGCTAC纯合子的HCC各16例,应用AffymetrixHuman Genome U133表达谱芯片进行分析,SAM分析(Significant Analysis ofMicroarray)两组表达谱差异,并聚类分析后,Real-time PCR法进行验证;在此分析基础上,另外分别选择GCGGGT纯合子、AGCTAC纯合子、GCGGGT/AGCTAC杂合子、AGCTAC/AGCTGC杂合子、GCGGAT/AGCTAC杂合子HCC各4例,行聚类分析验证等。
结果:我们对GCGGGT和AGCTAC纯合子HCC各16例行SAM分析发现41个差异表达基因(包括2个转录本),其中以IL-8在GCGGGT上调表达8倍;聚类分析发现该41个差异表达基因(转录本)可以将两组很好地区分开来。同时应用该41个差异表达基因(转录本)对后续20例HCC聚类分析发现可以将GCGGGT纯合子、AGCTAC纯合子、GCGGGT/AGCTAC杂合子、AGCTAC/AGCTGC杂合子、GCGGAT/AGCTAC杂合子等五组HCC很好地区分为两大组:即GCGGGT纯合子+GCGGGT/AGCTAC杂合子+GCGGAT/AGCTAC杂合子和AGCTAC纯合子+AGCTAC/AGCTGC杂合子。同时对总52例HCC聚类分析见相似结果。52例中临床表型分析发现前组的28例中有19例具有高侵袭转移潜能,而后组24例中有8例具有高侵袭转移潜能(P=0.025)。Real-time PCR和RT-PCR均验证了上述差异表达。
结论:不同HTPAP单体型的HCC具有不同的基因表达谱,这些在不同HTPAP单体型HCC中具有显著差异表达的基因可能于HTPAP基因有关,并影响肝癌的不同临床表型,改变其侵袭转移潜能,提示HCC中HTPAP的不同单体型可能是潜在侵袭转移相关的预测指标。
四、不同SNP基因型和单体型HTPAP蛋白在肝癌中的表达及其与术后无瘤生存(术后复发)的关系
目的:检测不同单体型的HCC中HTPAP蛋白的表达,探讨两者间的关系;检测665例HCC的6个SNPs位点,探讨SNPs和单体型与预后的关系。
方法:(1)应用免疫组织化学染色方法,观察HTPAP蛋白在377例HCC中的表达,分析SNPs和单体型与蛋白表达的关系;(2)显微切割2002—2003年手术切除的具有完整术后随访资料的665例HCC的石蜡包埋的肿瘤组织样本,提取DNA,应用焦磷酸测序方法对6个SNPs进行检测、单体型构建后,并统计665例术后无瘤生存时间(DFS,Disease-free Survival),探讨SNPs基因型、单体型与术后DFS(术后复发)的关系。
结果:(1)HTPAP蛋白的表达在377例HCC中有177例阳性染色(+～+++),占46.9%。染色特点与肝癌侵袭转移潜能(有无肝内播散灶和脉管癌栓)分析发现,377例中193例具有高侵袭转移潜能的HCC中有80例HTPAP染色为阳性(41.5%),而184例低高侵袭转移潜能的HCC中有97例为HTPAP染色阳性(52.7%)(P=0.028),提示HCC的侵袭转移潜能与HTPAP蛋白表达负相关。TagSNP+357C/G基因型与HTPAP染色发现GG+GC基因型的HCC较CC型的HTPAP蛋白表达低(P=0.002);而TagSNP+1838A/G分析发现GG+GA基因型的HCC较AA型的HTPAP蛋白表达无统计差异低(P=0.130)。单体型分析发现GCGGGT+GCGGAT单体型HCC较其它单体型的HTPAP表达亦显著降低(P=0.001),免疫组化染色分析提示不同的单体型能影响HTPAP蛋白的表达水平。
Kaplan-Meier生存曲线分析提示:TagSNP+357GC的Genotype为GG,GC,CC的HCC之间的术后无瘤生存时间具有统计差异,Genotype为CC的HCC术后无瘤生存较为GG和GC明显好(P＜0.001);而TagSNP+1838A/G的Genotype为GG,GA,AA的HCC之间的术后无瘤生存时间无统计差异(P=0.282)。单体型与预后分析发现GCGGGT纯合子较GCGGGT杂合子和其它单体型术后无瘤生存差(P＜0.001),提示具有较高的术后复发风险。Cox风险比例模型多因素分析发现TagSNP+357 GG+GC基因型和GCGGGT纯合子或杂合子单体型是术后无瘤生存(术后复发)差的独立相关因子(分别为GG:P=0.021,HR(HazardRatios)=1.51,95%C.I.=1.07-2.15,GC:P=0.030,HR=1.29,95%CI=1.03-1.62;GCGGGT纯合子:P=0.009,HR=1.59,95%C.I.=1.12-2.27,GCGGGT杂合子:P=0.022,HR=1.32,95%C.I.=1.04-1.67)。
结论:我们研究表明肿瘤转移抑制基因HTPAP上的SNPs会改变肿瘤的侵袭转移潜能。(1)Tag+357 GG+GC基因型和GCGGGT和GCGGAT单体型的HTPAP蛋白表达较+357CC和AGCTAC等其它单体型显著降低。(2)TagSNP+357 GG+GC基因型和GCGGGT纯合子或杂合子单体型是HCC术后无瘤生存差相关的独立因素,为HCC术后高复发风险的独立相关因子。
Hepatocellular carcinoma(HCC) is one of the most common and aggressive malignancies worldwide.It has been ranked the third in terms of cancer mortality worldwide and the 2nd cancer killer in China since 1990s.Despite clinical advances in surgical resection,the prognosis of HCC still remains dismal.Metastatic recurrence after HCC resection is one of the major obstacles to prolonging survival.Therefore, there is a tremendous interest and urgency to search for molecules related to HCC metastasis that would provide new predictors for HCC metastasis as well as new targets for intervention.
HTPAP is a newly-discovered tumor-related gene,its official symbol is PPAPDC1B(Phosphatidic acid phosphatase type 2 domain containing 1B).It is mapped on chorosome 8p12 with seven exons and eight introns.Its full length is 4459bp with a cDNA of 826bp encoding 175 amino acid protein.The abnormal expression of HTPAP has been found in several human tumors,and has been showed a potential functional role of inhibiting the proliferation,invasion and metastasis of cancer cells.In our previous study,it has been demonstrated both in vitro and in vivo that HTPAP could suppress the invasion and metastasis of HCC,which indicate that HTPAP might be a new metastatic suppressor.
In our previous study on the polymorphisms of HTPAP in HCC,we found allele C to G change on the rs1149.The risk associated with GG+GC genotype(61%) and G allele(39%) at rs1149 in HCCs with intrahepatic metastasis and vascular invasion is high than the without(39%,24%respectively)(P=0.046,P=0.026 respectively).In HCC,the genotype GG+GC was correlated with tumor number(P=0.026),TNM stage(P=0.026),5-year disease-free survival(DFS)(P=0.005),and 5-year overall survival(OS)(P=0.014).The allele C to G change at rs1149 might have an effect on HTPAP expression level and protein function and structure,consequently conferring up-regulating metastasis potential.
Based on the above findings,we conducted the following study on the polymorphisms of HTPAP in HCC tissues using laser captured tissue microdissection and single nucleotide polymorphisms(SNPs) analysis.We sequence across the HTPAP region and detect all the SNPs,and evaluate the associations of SNP genotypes and haplotypes with metastasis potential,postoperative disease-free survival(HCC recurrence),and investigate the possible mechanisms.
PARTⅠReconstruction of the haplotypes for HTPAP gene and their association with the metastatic potential of hepatocellular carcinoma.
Objective:In our previous studies,HTPAP was found to be a candidate metastasis suppressor gene for HCC.In this study,we want to analyze the haplotypes of HTPAP through identifying SNPs on it,and their relations to invasion ability of HCC.
Methods:We detected SNPs by direct sequencing of 7.5kb including HTPAP(4.5kb) and its 3',5'-flanking region(3kb),and then analyzed genotypes of the SNPs in DNA samples from 864 cases of microdiessected fresh HCC tissues.The 864 cases were enrolled to haplotype reconstruction by PHASE software.We calculate the genotypes, haplotypes and their relations to metastatic potential of hepatocellular carcinoma.
Results:Six SNPs were detected(-1053 A/G,+64G/C,+357C/G(also as rs1149), +1648-/TAAG(we renamed the deletion genotype as T,and insertion genotype as G, as follows),+1838A/G,+3528C/T).A total of 12 haplotypes were found,four of them (A-G-C-T-A-C,G-C-G-G-G-T,A-G-C-T-G-C,G-C-G-G-A-T) occurred most frequently(94.62%),and accounted for 793 cases(91.78%).Five SNPs were found to be in a strong linkage disequilibrium(LD)(r~2=0.85-1.0),and their association between them can then be explained by tag SNP+357C/G..The other one at +1838A/G had a historical recombination,and were correlated with them with r~2 range of 0.32-0.36. Association analysis revealed that carriers of alleles G at tag SNP +357C/G.increased higher metastatic potential risk in a dose-dependent manner with a OR of 1.77 (95%C.I.=1.32-2.36,P=0.0001)from CC to GC,and a OR of 2.11(95%C.I.= 1.28-3.48,P=0.003) from CC to GG.No significance was found between +1838A/G genotype and higher metastatic potential risk(GA:OR=1.09(0.81-1.46),P=0.564,: GG:OR=1.39(0.91-2.12),P=0.126).The haplotype G-C-G-G-G-T were significantly associated with higher metastatic potential risk(OR=1.50(1.18-1.91),P=0.001),but none with haplotypes of G-C-G-G-A-T,A-G-C-T-A-C and A-G-C-T-G-C.
Conclusions:The tag SNP+357 GG+GC genotype and.haplotype G-C-G-G-G-T were significantly associated with higher metastatic potential risk in these Chinese HCC patients,and the functional analysis of these different genotypes and haplotypes of HTPAP might provide a new way in the prediction of patients' prognosis and tumor recurrence.
PARTⅡThe mechanism of different genotypes and haplotypes of HTPAP involved in the regulation of HCC metastasis potential
1.HTPAP expression and its association with haplotypcs of HTPAP
Objective:Evaluating the effect of haplotypes on mRNA expression.
Methods:After construction of pEGFP-HTPAP the recombinated plasmids,we evaluate the HTPAP expression level(its three known isoforms) in HCCs composed of four common HTPAP haplotype A-G-C-T-A-C,G-C-G-G-G-T,A-G-C-T-G-C and G-C-G-G-A-T by quantitive real-time PCR.
Result:We constructed the recombinated plasmids successfully.HTPAP expression in 170 HCC cases were calculated by quantitive real-time PCR.Among them,21cases belonged to GCGGGT homozygotes carriers,65 AGCTAC homozygotes,52 AGCTAC/GCGGGT heterozygotes,13 AGCTAC/GCGGAT heterozygotes and 19 AGCTAC/AGCTGC heterozygotes.The expression levels in groups of AGCTAC homozygotes and AGCTAC/AGCTGC heterozygotes were higher than those of GCGGGT homozygotes,AGCTAC/GCGGGT and 13 AGCTAC/GCGGAT heterozygotes.Overally,no significance was found between them(P＞0.05).
Conclusions:Different HTPAP haplotypes can influence the expression level,but none of them reach significance.
2.HTPAP promoter haplotypes and their transcriptional activities
Objective:Evaluating the effect of three HTPAP promoter haplotypes on their transcriptional activities.
Methods:We generated the three HTPAP promoter haplotype luciferase reporter vectors(pGL3-basic) spanning from-1764 to +315 bp of the HTPAP promoter region, and transiently transfect these promoters into MHCC-97H,HepG2 and Hela cells,and detect the luciferase index.
Result:Compared with void pGL3-basic vectors,three recombinated promoter plasmids observed obvious transcriptional activity.-1053AA/+64GG haplotype promoter manifested significantly higher luciferase index than that of -1053 AG/+64 GC and -1053 GG/+64 CC promoter(P=0.005,0.009 respectively).However,no significance was observed between -1053AG/+64 GC and -1053 GG/+64 CC promoter(P=0.26)..
Conclusions:The different haplotype promoters could affect the transcriptional activity signficantly.
3 The investigation of mechanism regulating the metastasis potential with different HTPAP genotype and haplotype by Affymetrix gene-expression biochip
Objective:To analyse the different expression pattern between HCC of different HTPAP haplotype,and investigate possible mechanism regulating the metastasis potential.
Methods:We randomly selected 16 HCCs in GCGGGT homozygote and AGCTAC homozygote each for genechip analysis by Affymetrix Human Genome U133.By SAM(Significant Analysis of Microarray),we analysed the different expression between them,and reevaluate.the different expression by Real-time PCR.To broaden our research,we again randomly selected 4 cases in each group(AGCTAC/GCGGGT heterozygotes,AGCTAC/GCGGAT heterozygotes,AGCTAC/AGCTGC heterozygotes,GCGGGT homozygote and AGCTAC homozygote) for further SAM and Cluster&TreeView analysis.
Result:By SAM analysis(Significant Analysis of Microarray),we obtained 41significantly different genes(including 2 transcripts)(P＜0.05,fold change＞2) between GCGGGT and AGCTAC homozygote carriers(the whole 41 genes or transcripts were overexpressed in GCGGGT homozygote carriers).By uncentered correlation and average linkage analysis,we accurately classified 16 GCGGGT homozygotes tumors(88%,with 2 sample missed) from 16 AGCTAC homozygotes with the above 41 significant genes(transcripts) in the classifier.Further evaluation of the classifier in randomly selected 4 cases in each group(AGCTAC/GCGGGT heterozygotes carrier,AGCTAC/GCGGAT heterozygotes carrier, AGCTAC/AGCTGC heterozygotes carrier,GCGGGT homozygotes carrier and AGCTAC homozygotes carrier) showed the five groups of HCC could be distinguish correctly into two large groups(groupⅠ:GCGGGT homozygotes, AGCTAC/GCGGGT heterozygotes,AGCTAC/GCGGAT heterozygotes,and groupⅡ:AGCTAC homozygotes and AGCTAC/AGCTGC heterozygotes)(83%,with 2 sample missed).When we applied the classifier in the all 52 HCCs,we found the 52 HCC samples could also be distinguish correctly into two large groups as above (92%,with only two missed).IL-8(with fold change over 8) and TLR-2(over 3) were the most overexpressed in GCGGGT homozygote HCC group in the classifier.By RT-PCR and real-time quantitative PCR,we found IL-8 and TLR-2 were overexpressed in tumors of GCGGGT homozygotes,AGCTAC/GCGGGT heterozygotes,and AGCTAC/GCGGAT heterozygotes more than in those of AGCTAC/AGCTGC heterozygotes and AGCTAC homozygotes.
When we study the phenotype in the 52 HCC patients,19 of 28 patients in groupⅠand 8 out of 24 in groupⅡwere found high metastasis potential(P=0.025),indicating different haplotype carriers might contribute different risk of metastatic potential.
Conclusions:The different expression patterns might occurred between different HTPAP haplotype HCC.These significantly different genes could functionally associated with different HTPAP haplotype,contributedly rendered changes in HCC phenotype such as invasion ability and metastasis potential,indicating that the different HTPAP haplotypes might be the prognostic predicator of HCC metastasis potential.
4 The association of HTPAP genotype and haplotype with protein expression and postoperative disease-free survival in HCC
Objective:We evaluate the relationship between the protein expression and HTPAP genotype and haplotype by immuno-histochemical analysis on expression of HTPAP in paraffin enbedded HCC tissues.Also,we investigate the association between postoperative disease-free survival(DFS)(HCC recurrence) and HTPAP genotype and haplotype by detecting 665 cases of HCC with 5-year follow-up.
Methods:By immuno-histochemical analysis,we observed the expression of HTPAP in 377 cases,and evaluate the relationship between the protein expression and HTPAP genotype and haplotype.665 cases of HCC from Jan.2002 to June 2003 were enrolled for examining the SNPs by prosequencing 96,the association between postoperative DFS and HTPAP genotype and haplotype were investigated.
Results:By immuno-histochemical analysis in 377 cases of HCC,we observed the positive staining in 177 case(46.9%).Overally,two patterns of positive immunohistochemical staining have been occurred in cytoplasm.We observed intensified granular staining in cancer cells of the whole nodules,while the scattered positive staining of HCC in cancer nodules was also frequently present.Meanwhile, we found strong HTPAP staining in hyperplastic bile duct epithelial cell,but no positive staining in normal hepatic cells,liver cirrhotic cells,and infiltrating lymphocyte.Positive protein expression occurred more frequently in HCC cases with high metastasis potential compared with that with low potential(P=0.028).When we analysed the association of HTPAP genotypes and haplotypes with protein expression, we found the tag SNP +357(GG+GC) genotype and haplotypes(GCGGGT+GCGGAT) were negatively associated with HTPAP expression(P=0.002,P=0.001,respectively).No significance of HTPAP staining was found between tag SNP+1838(AG+GG) and AA genotype HCCs(P=0.130).
Kaplan-Meier analysis showed that the tag SNP+357 GG and GC was significantly associated with a decreased probability of postoperative DFS compared with +357CC genotype(P＜0.001),but we found no significance of postoperative DFS between the tagSNP+1838 GG,AG and AA genotype HCCs(P=0.282).We found the poorer postoperative DFS in HCCs of GCGGGT homozygotes and heterozygotes than that of others(P＜0.001),showing that GCGGGT homozygotes or heterozygotes was associated with a decreased probability of postoperative DFS compared with other haplotypes.
We recalculated the HRs for each tag SNP+357G/C genotyoes,and for haplotype of GCGGGT homozygotes,heterozygotes and others/others following adjustments for clinical characteristics.This analysis showed that only the +357 GG+GC genotype and GCGGGT homozygotes and heterozygotes were negatively associated with postoperative DFS(for+357GG genotype,P=0.021,HR(Hazard Ratios)=1.51, 95%C.I.=1.07-2.15,for+357GC genotype,P=0.030,HR=1.29,95%CI=1.03-1.62; for GCGGGT homozygotes,P=0.009,HR=1.59,95%C.I.=1.12-2.27,and for GCGGGT heterozygotes,P=0.022,HR=1.32,95%C.I.=1.04-1.67).
Conclusions:Our results show that variants at tumor suppressor have different effects on HCC metastasis potential.The tag SNP +357(GG+GC) genotype and haplotypes(GCGGGT+GCGGAT) were negatively associated with HTPAP expression. +357 GG+GC genotype and GCGGGT homozygote and heterozygotes of HTPAP are independently associated with a poor postoperative DFS in HCC patients after surgical resection,which indicates that +357 GG+GC genotype and GCGGGT homozygotes and heterozygotes might have great value in prediction of HCC metastasis potential(postoperative recurrence) and prognosis..
Conclusions
1.The tag SNP +357 GG+GC genotype and.haplotype G-C-G-G-G-T were significantly associated with higher metastatic potential risk in these Chinese HCC patients.
2.Different HTPAP haplotypes can influence the expression level,but none of them reach significance.The SNP genotype and haplotype in promoter could affect the transcriptional activity signficantly.
3.The different expression patterns might occurred between different HTPAP haplotype HCC associated with different HTPAP haplotype,and contributedly render changes in HCC phenotype such as invasion ability and metastasis potential indicating that the different HTPAP haplotypes could be the prognostic predicator of HCC metastasis potential.
4.Our results show that variants at tumor suppressor have different effects on HCC metastasis potential.+357GG+GC genotype and GCGGGT homozygote and heterozygotes are independently associated with a poor prognosis in HCC patients after surgical resection,which indicates they might have great value in prediction of HCC metastasis potential and prognosis
Potential application of this study
1.The tag SNP +357 GG+GC genotype and.haplotype G-C-G-G-G-T were significantly associated with higher metastatic potential risk,and might be a new biomarker of tumor metastasis.
2.Genotype +357 GG+GC and GCGGGT homozygote and heterozygotes are independently associated with a poor prognosis in HCC patients after surgical resection,which indicates they might have the potential of being a new genetic marker in prediction of metastasis and prognosis
Novelties
1.To the best of our knowledge,this is the first report investigating the association between the variants at HTPAP gene and HCC metastasis potential.
2.To the best of our knowledge,this is the first time to analyse different expression pattern between different HTPAP haplotype carriers in HCC by global gene-expression array to investigate the mechanism involved in the regulation of metastasis.
3.In addition,our data for the first time show that Genotype +357 GG+GC and GCGGGT homozygote and heterozygotes of HTPAP are independently associated with a decreased probability of postoperative DFS in HCC patients.
HTPAP基因是肿瘤相关基因,其正式名为PPAPDC1B(Phosphatidic acidphosphatase type 2 domain containing 1B),含有1B功能域的2型磷脂酸磷酸酯酶。该基因定位于8p12,含有7个外显子、6个内含子。基因全长4459 bp,cDNA长为826 bp,编码一个具有175个氨基酸的蛋白质。已有研究发现一些人类肿瘤中该基因的异常转录表达,提示该基因具有抑制肿瘤细胞增殖、降低侵袭性、抑制肿瘤转移的作用。我们前期经体内、体外研究初步证实该基因可抑制HCC的侵袭和转移,提示HTPAP基因是一新的HCC转移抑制基因。
前期研究中,我们初步通过对肝癌病人肿瘤组织HTPAP基因的单核苷酸多态性分析研究发现,HCC病人HTPAP基因上的rs1149位点存在C→G多态性。伴有肝内播散和/或脉管侵犯的病人血浆循环DNA rs1149位点GG+GC基因型频率和G等位基因频率分别为61%和39%,明显高于不伴有肝内播散和脉管侵犯的病人该位点GG+GC基因型频率和G等位基因频率(分别为39%和24%),二组差异显著(P=0.046,P=0.026)。rs1149位点GG+GC基因型与肿瘤数目P=0.026),TNM分期(P=0.026),病人无瘤生存率(P=0.005)和总体生存率(P=0.014)均密切相关。rs1149位点上的C→G多态性可能影响HTPAP基因的表达或引起基因编码的蛋白质及其功能的改变,从而导致HCC病人肿瘤侵袭性的增高。
基于上述理论和实验基础,本实验拟应用激光捕获组织显微切割获取纯肝癌组织DNA,对HTPAP基因的SNP位点进行检测并行单体型构建,探讨HTPAP基因的SNP、单体型与肝癌转移潜能的关系及其相关机制,以及与肝癌病人转移复发和预后的关系。
第一部分肿瘤抑制基因HTPAP的SNP检测、单体型构建及其与肝癌侵袭性能的关系探讨
目的:在前期研究基础上通过对大样本的HCC病人肿瘤组织DNA的HTPAP基因的单核苷酸多态性(single nucleotide polymorphisms,SNPs)检测、单体型构建后,探讨其与肝癌侵袭性关系。
方法:应用激光捕获组织显微切割从864例HCC手术样本中获取相对纯的HCC细胞、提取DNA,对30例HCC的HTPAP基因全长和5'、3'-flanking region(侧翼区)的3kb启动子区域进行ABI仪测序发现SNP位点后,应用焦磷酸测序仪行864例样本SNP检测,并予单体型构建,分析其与肝癌侵袭转移潜能(有无肝内播散,脉管癌栓)的关系。
结果:在7.5 Kb测序区域中,共发现6个SNP位点[-1053 A/G、+64G/C、+357C/G、+1648-/TAAG(无插入和插入TAAG等位基因分别以T/G示,后同)、+1838A/G、+3528C/T]。单体型构建研究发现HTPAP中共存在12种单体型,其中A-G-C-T-A-C,G-C-G-G-G-T,A-G-C-T-G-C,G-C-G-G-A-T(依次为6个SNP的等位基因:+3528C/T—+1838A/G—+1648-/TAAG—+357C/G—+64G/C—-1053A/G,后同)四种单体型占94.62%。793例(占91.78%)HCC由该4种单体型构成。5个SNP(-1053 A/G、+64G/C、+357C/G、+1648-/TAAG、+3528C/T)表现为强连锁不平衡关系(r~2=0.85-1.0),而+1838A/G出现重组现象,与上述5个SNP连锁较弱(r~2=0.32-0.36);临床病理特征分析提示SNP+357C/G的基因型GG+GC与HCC高侵袭转移潜能正相关[GC:OR=1.77,95%C.I.=1.32-2.36,P=0.0001;GG:OR=2.11(1.28-3.48),P=0.003],呈等位基因G剂量依存性。而+1838A/G虽然处于外显子之中,但是与HCC侵袭转移潜能并无相关[GA:OR=1.09(0.81-1.46),P=0.564;GG:OR=1.39(0.91-2.12),P=0.126]。单体型G-C-G-G-G-T与高侵袭转移潜能正相关[OR=1.50(1.18-1.91),P=0.001],而G-C-G-G-A-T,A-G-C-T-A-C和A-G-C-T-G-C则与HCC侵袭性无关。
结论:肿瘤转移抑制基因HTPAP的TagSNP+357 GG+GC基因型和G-C-G-G-G-T单体型与HCC高侵袭转移潜能正相关,针对不同SNP基因型和单体型分析可能会为肝癌转移复发、预后预测提供新的途径和分子遗传学指标。
第二部分HTPAP基因的SNP基因型和单体型影响肝癌侵袭转移潜能的相关机制研究及其对预后的影响
一、HCC中HTPAP单体型与基因表达关系
目的:检测HCC中HTPAP基因的表达,探讨HTPAP基因的TagSNP+357C/G基因型和相关的单体型对与基因表达的的影响。
方法:(1)构建pEGFP-HTPAP载体真核表达质粒,应用绝对定量PCR(quantitative real-time PCR)检测HCC中HTPAP RNA的表达(含3种同素异构体).(2)随机选取170例以A-G-C-T-A-C,G-C-G-G-G-T,A-G-C-T-G-C和G-C-G-G-A-T等四种HTPAP基因主要单体型组成的HCC组织,并提取总RNA。
结果:成功构建pEGFP-HTPAP载体真核表达质粒。对4种单体型组成的170例HCC进行HTPAP基因表达水平的定量分析,其中GCGGGT纯合子21例、AGCTAC纯合子65例、AGCTAC/GCGGGT杂合子52例、AGCTAC/GCGGAT杂合子13例、AGCTAC/AGCTGC杂合子19例。发现HCC中HTPAP基因表达在AGCTAC纯合子和AGCTAC/AGCTGC杂合子病人较GCGGGT纯合子、AGCTAC/GCGGGT杂合子和AGCTAC/GCGGAT杂合子表达升高,但未达到统计意义(P＞0.05)。
结论:HCC中不同HTPAP单体型能影响HTPAP表达水平,但是无统计学差异。
二、HCC中HTPAP基因启动子单体型与转录活性的关系
目的:通过构建HTPAP的3个启动子单体型,探讨不同HTPAP启动子单体型对基因转录活性的影响。
方法:PCR方法克隆出跨越基因上游5'-flanking的-1764bp至基因内部+315bp的启动子序列(2079bp)。该区域含-1053AG和+64GC两个SNP,由于存在完全LD,故只有三种单体型(-1053 AA/+64GG、-1053 AG/+64 GC和-1053 GG/+64 CC),上述HTPAP启动子单体型pGL3质粒,并分别转染MHCC-97H、HepG2肝癌细胞和Hela细胞,采用双荧光报告法,检测荧光素酶的活性,重复3次。
结果:三种单体型的HTPAP启动子都具有显著转录活性(与空pGL3-basic质粒相比),其中-1053 AA/+64GG单体型启动子转录活性明显强于-1053 GG/+64 CC(P=0.005)和-1053 AG/+64 GC单体型启动子(P=0.009)。而-1053GG/+64 CC和-1053 AG/+64 GC启动子单体型间未见明显差异(P=0.26)。
结论:HTPAP启动子SNP(-1053AG和+64GC)的不同单体型对基因的转录活性有显著影响,能启动不同水平的基因转录。
三、利用基因芯片研究HTPAP单体型调控HCC侵袭转移潜能的调控机制
目的:应用表达谱芯片分析不同单体型HCC的差异表达,探不同单体型HTPAP基因影响HCC的侵袭性潜能可能机制。
方法:选择GCGGGT和AGCTAC纯合子的HCC各16例,应用AffymetrixHuman Genome U133表达谱芯片进行分析,SAM分析(Significant Analysis ofMicroarray)两组表达谱差异,并聚类分析后,Real-time PCR法进行验证;在此分析基础上,另外分别选择GCGGGT纯合子、AGCTAC纯合子、GCGGGT/AGCTAC杂合子、AGCTAC/AGCTGC杂合子、GCGGAT/AGCTAC杂合子HCC各4例,行聚类分析验证等。
结果:我们对GCGGGT和AGCTAC纯合子HCC各16例行SAM分析发现41个差异表达基因(包括2个转录本),其中以IL-8在GCGGGT上调表达8倍;聚类分析发现该41个差异表达基因(转录本)可以将两组很好地区分开来。同时应用该41个差异表达基因(转录本)对后续20例HCC聚类分析发现可以将GCGGGT纯合子、AGCTAC纯合子、GCGGGT/AGCTAC杂合子、AGCTAC/AGCTGC杂合子、GCGGAT/AGCTAC杂合子等五组HCC很好地区分为两大组:即GCGGGT纯合子+GCGGGT/AGCTAC杂合子+GCGGAT/AGCTAC杂合子和AGCTAC纯合子+AGCTAC/AGCTGC杂合子。同时对总52例HCC聚类分析见相似结果。52例中临床表型分析发现前组的28例中有19例具有高侵袭转移潜能,而后组24例中有8例具有高侵袭转移潜能(P=0.025)。Real-time PCR和RT-PCR均验证了上述差异表达。
结论:不同HTPAP单体型的HCC具有不同的基因表达谱,这些在不同HTPAP单体型HCC中具有显著差异表达的基因可能于HTPAP基因有关,并影响肝癌的不同临床表型,改变其侵袭转移潜能,提示HCC中HTPAP的不同单体型可能是潜在侵袭转移相关的预测指标。
四、不同SNP基因型和单体型HTPAP蛋白在肝癌中的表达及其与术后无瘤生存(术后复发)的关系
目的:检测不同单体型的HCC中HTPAP蛋白的表达,探讨两者间的关系;检测665例HCC的6个SNPs位点,探讨SNPs和单体型与预后的关系。
方法:(1)应用免疫组织化学染色方法,观察HTPAP蛋白在377例HCC中的表达,分析SNPs和单体型与蛋白表达的关系;(2)显微切割2002—2003年手术切除的具有完整术后随访资料的665例HCC的石蜡包埋的肿瘤组织样本,提取DNA,应用焦磷酸测序方法对6个SNPs进行检测、单体型构建后,并统计665例术后无瘤生存时间(DFS,Disease-free Survival),探讨SNPs基因型、单体型与术后DFS(术后复发)的关系。
结果:(1)HTPAP蛋白的表达在377例HCC中有177例阳性染色(+～+++),占46.9%。染色特点与肝癌侵袭转移潜能(有无肝内播散灶和脉管癌栓)分析发现,377例中193例具有高侵袭转移潜能的HCC中有80例HTPAP染色为阳性(41.5%),而184例低高侵袭转移潜能的HCC中有97例为HTPAP染色阳性(52.7%)(P=0.028),提示HCC的侵袭转移潜能与HTPAP蛋白表达负相关。TagSNP+357C/G基因型与HTPAP染色发现GG+GC基因型的HCC较CC型的HTPAP蛋白表达低(P=0.002);而TagSNP+1838A/G分析发现GG+GA基因型的HCC较AA型的HTPAP蛋白表达无统计差异低(P=0.130)。单体型分析发现GCGGGT+GCGGAT单体型HCC较其它单体型的HTPAP表达亦显著降低(P=0.001),免疫组化染色分析提示不同的单体型能影响HTPAP蛋白的表达水平。
Kaplan-Meier生存曲线分析提示:TagSNP+357GC的Genotype为GG,GC,CC的HCC之间的术后无瘤生存时间具有统计差异,Genotype为CC的HCC术后无瘤生存较为GG和GC明显好(P＜0.001);而TagSNP+1838A/G的Genotype为GG,GA,AA的HCC之间的术后无瘤生存时间无统计差异(P=0.282)。单体型与预后分析发现GCGGGT纯合子较GCGGGT杂合子和其它单体型术后无瘤生存差(P＜0.001),提示具有较高的术后复发风险。Cox风险比例模型多因素分析发现TagSNP+357 GG+GC基因型和GCGGGT纯合子或杂合子单体型是术后无瘤生存(术后复发)差的独立相关因子(分别为GG:P=0.021,HR(HazardRatios)=1.51,95%C.I.=1.07-2.15,GC:P=0.030,HR=1.29,95%CI=1.03-1.62;GCGGGT纯合子:P=0.009,HR=1.59,95%C.I.=1.12-2.27,GCGGGT杂合子:P=0.022,HR=1.32,95%C.I.=1.04-1.67)。
结论:我们研究表明肿瘤转移抑制基因HTPAP上的SNPs会改变肿瘤的侵袭转移潜能。(1)Tag+357 GG+GC基因型和GCGGGT和GCGGAT单体型的HTPAP蛋白表达较+357CC和AGCTAC等其它单体型显著降低。(2)TagSNP+357 GG+GC基因型和GCGGGT纯合子或杂合子单体型是HCC术后无瘤生存差相关的独立因素,为HCC术后高复发风险的独立相关因子。
Hepatocellular carcinoma(HCC) is one of the most common and aggressive malignancies worldwide.It has been ranked the third in terms of cancer mortality worldwide and the 2nd cancer killer in China since 1990s.Despite clinical advances in surgical resection,the prognosis of HCC still remains dismal.Metastatic recurrence after HCC resection is one of the major obstacles to prolonging survival.Therefore, there is a tremendous interest and urgency to search for molecules related to HCC metastasis that would provide new predictors for HCC metastasis as well as new targets for intervention.
HTPAP is a newly-discovered tumor-related gene,its official symbol is PPAPDC1B(Phosphatidic acid phosphatase type 2 domain containing 1B).It is mapped on chorosome 8p12 with seven exons and eight introns.Its full length is 4459bp with a cDNA of 826bp encoding 175 amino acid protein.The abnormal expression of HTPAP has been found in several human tumors,and has been showed a potential functional role of inhibiting the proliferation,invasion and metastasis of cancer cells.In our previous study,it has been demonstrated both in vitro and in vivo that HTPAP could suppress the invasion and metastasis of HCC,which indicate that HTPAP might be a new metastatic suppressor.
In our previous study on the polymorphisms of HTPAP in HCC,we found allele C to G change on the rs1149.The risk associated with GG+GC genotype(61%) and G allele(39%) at rs1149 in HCCs with intrahepatic metastasis and vascular invasion is high than the without(39%,24%respectively)(P=0.046,P=0.026 respectively).In HCC,the genotype GG+GC was correlated with tumor number(P=0.026),TNM stage(P=0.026),5-year disease-free survival(DFS)(P=0.005),and 5-year overall survival(OS)(P=0.014).The allele C to G change at rs1149 might have an effect on HTPAP expression level and protein function and structure,consequently conferring up-regulating metastasis potential.
Based on the above findings,we conducted the following study on the polymorphisms of HTPAP in HCC tissues using laser captured tissue microdissection and single nucleotide polymorphisms(SNPs) analysis.We sequence across the HTPAP region and detect all the SNPs,and evaluate the associations of SNP genotypes and haplotypes with metastasis potential,postoperative disease-free survival(HCC recurrence),and investigate the possible mechanisms.
PARTⅠReconstruction of the haplotypes for HTPAP gene and their association with the metastatic potential of hepatocellular carcinoma.
Objective:In our previous studies,HTPAP was found to be a candidate metastasis suppressor gene for HCC.In this study,we want to analyze the haplotypes of HTPAP through identifying SNPs on it,and their relations to invasion ability of HCC.
Methods:We detected SNPs by direct sequencing of 7.5kb including HTPAP(4.5kb) and its 3',5'-flanking region(3kb),and then analyzed genotypes of the SNPs in DNA samples from 864 cases of microdiessected fresh HCC tissues.The 864 cases were enrolled to haplotype reconstruction by PHASE software.We calculate the genotypes, haplotypes and their relations to metastatic potential of hepatocellular carcinoma.
Results:Six SNPs were detected(-1053 A/G,+64G/C,+357C/G(also as rs1149), +1648-/TAAG(we renamed the deletion genotype as T,and insertion genotype as G, as follows),+1838A/G,+3528C/T).A total of 12 haplotypes were found,four of them (A-G-C-T-A-C,G-C-G-G-G-T,A-G-C-T-G-C,G-C-G-G-A-T) occurred most frequently(94.62%),and accounted for 793 cases(91.78%).Five SNPs were found to be in a strong linkage disequilibrium(LD)(r~2=0.85-1.0),and their association between them can then be explained by tag SNP+357C/G..The other one at +1838A/G had a historical recombination,and were correlated with them with r~2 range of 0.32-0.36. Association analysis revealed that carriers of alleles G at tag SNP +357C/G.increased higher metastatic potential risk in a dose-dependent manner with a OR of 1.77 (95%C.I.=1.32-2.36,P=0.0001)from CC to GC,and a OR of 2.11(95%C.I.= 1.28-3.48,P=0.003) from CC to GG.No significance was found between +1838A/G genotype and higher metastatic potential risk(GA:OR=1.09(0.81-1.46),P=0.564,: GG:OR=1.39(0.91-2.12),P=0.126).The haplotype G-C-G-G-G-T were significantly associated with higher metastatic potential risk(OR=1.50(1.18-1.91),P=0.001),but none with haplotypes of G-C-G-G-A-T,A-G-C-T-A-C and A-G-C-T-G-C.
Conclusions:The tag SNP+357 GG+GC genotype and.haplotype G-C-G-G-G-T were significantly associated with higher metastatic potential risk in these Chinese HCC patients,and the functional analysis of these different genotypes and haplotypes of HTPAP might provide a new way in the prediction of patients' prognosis and tumor recurrence.
PARTⅡThe mechanism of different genotypes and haplotypes of HTPAP involved in the regulation of HCC metastasis potential
1.HTPAP expression and its association with haplotypcs of HTPAP
Objective:Evaluating the effect of haplotypes on mRNA expression.
Methods:After construction of pEGFP-HTPAP the recombinated plasmids,we evaluate the HTPAP expression level(its three known isoforms) in HCCs composed of four common HTPAP haplotype A-G-C-T-A-C,G-C-G-G-G-T,A-G-C-T-G-C and G-C-G-G-A-T by quantitive real-time PCR.
Result:We constructed the recombinated plasmids successfully.HTPAP expression in 170 HCC cases were calculated by quantitive real-time PCR.Among them,21cases belonged to GCGGGT homozygotes carriers,65 AGCTAC homozygotes,52 AGCTAC/GCGGGT heterozygotes,13 AGCTAC/GCGGAT heterozygotes and 19 AGCTAC/AGCTGC heterozygotes.The expression levels in groups of AGCTAC homozygotes and AGCTAC/AGCTGC heterozygotes were higher than those of GCGGGT homozygotes,AGCTAC/GCGGGT and 13 AGCTAC/GCGGAT heterozygotes.Overally,no significance was found between them(P＞0.05).
Conclusions:Different HTPAP haplotypes can influence the expression level,but none of them reach significance.
2.HTPAP promoter haplotypes and their transcriptional activities
Objective:Evaluating the effect of three HTPAP promoter haplotypes on their transcriptional activities.
Methods:We generated the three HTPAP promoter haplotype luciferase reporter vectors(pGL3-basic) spanning from-1764 to +315 bp of the HTPAP promoter region, and transiently transfect these promoters into MHCC-97H,HepG2 and Hela cells,and detect the luciferase index.
Result:Compared with void pGL3-basic vectors,three recombinated promoter plasmids observed obvious transcriptional activity.-1053AA/+64GG haplotype promoter manifested significantly higher luciferase index than that of -1053 AG/+64 GC and -1053 GG/+64 CC promoter(P=0.005,0.009 respectively).However,no significance was observed between -1053AG/+64 GC and -1053 GG/+64 CC promoter(P=0.26)..
Conclusions:The different haplotype promoters could affect the transcriptional activity signficantly.
3 The investigation of mechanism regulating the metastasis potential with different HTPAP genotype and haplotype by Affymetrix gene-expression biochip
Objective:To analyse the different expression pattern between HCC of different HTPAP haplotype,and investigate possible mechanism regulating the metastasis potential.
Methods:We randomly selected 16 HCCs in GCGGGT homozygote and AGCTAC homozygote each for genechip analysis by Affymetrix Human Genome U133.By SAM(Significant Analysis of Microarray),we analysed the different expression between them,and reevaluate.the different expression by Real-time PCR.To broaden our research,we again randomly selected 4 cases in each group(AGCTAC/GCGGGT heterozygotes,AGCTAC/GCGGAT heterozygotes,AGCTAC/AGCTGC heterozygotes,GCGGGT homozygote and AGCTAC homozygote) for further SAM and Cluster&TreeView analysis.
Result:By SAM analysis(Significant Analysis of Microarray),we obtained 41significantly different genes(including 2 transcripts)(P＜0.05,fold change＞2) between GCGGGT and AGCTAC homozygote carriers(the whole 41 genes or transcripts were overexpressed in GCGGGT homozygote carriers).By uncentered correlation and average linkage analysis,we accurately classified 16 GCGGGT homozygotes tumors(88%,with 2 sample missed) from 16 AGCTAC homozygotes with the above 41 significant genes(transcripts) in the classifier.Further evaluation of the classifier in randomly selected 4 cases in each group(AGCTAC/GCGGGT heterozygotes carrier,AGCTAC/GCGGAT heterozygotes carrier, AGCTAC/AGCTGC heterozygotes carrier,GCGGGT homozygotes carrier and AGCTAC homozygotes carrier) showed the five groups of HCC could be distinguish correctly into two large groups(groupⅠ:GCGGGT homozygotes, AGCTAC/GCGGGT heterozygotes,AGCTAC/GCGGAT heterozygotes,and groupⅡ:AGCTAC homozygotes and AGCTAC/AGCTGC heterozygotes)(83%,with 2 sample missed).When we applied the classifier in the all 52 HCCs,we found the 52 HCC samples could also be distinguish correctly into two large groups as above (92%,with only two missed).IL-8(with fold change over 8) and TLR-2(over 3) were the most overexpressed in GCGGGT homozygote HCC group in the classifier.By RT-PCR and real-time quantitative PCR,we found IL-8 and TLR-2 were overexpressed in tumors of GCGGGT homozygotes,AGCTAC/GCGGGT heterozygotes,and AGCTAC/GCGGAT heterozygotes more than in those of AGCTAC/AGCTGC heterozygotes and AGCTAC homozygotes.
When we study the phenotype in the 52 HCC patients,19 of 28 patients in groupⅠand 8 out of 24 in groupⅡwere found high metastasis potential(P=0.025),indicating different haplotype carriers might contribute different risk of metastatic potential.
Conclusions:The different expression patterns might occurred between different HTPAP haplotype HCC.These significantly different genes could functionally associated with different HTPAP haplotype,contributedly rendered changes in HCC phenotype such as invasion ability and metastasis potential,indicating that the different HTPAP haplotypes might be the prognostic predicator of HCC metastasis potential.
4 The association of HTPAP genotype and haplotype with protein expression and postoperative disease-free survival in HCC
Objective:We evaluate the relationship between the protein expression and HTPAP genotype and haplotype by immuno-histochemical analysis on expression of HTPAP in paraffin enbedded HCC tissues.Also,we investigate the association between postoperative disease-free survival(DFS)(HCC recurrence) and HTPAP genotype and haplotype by detecting 665 cases of HCC with 5-year follow-up.
Methods:By immuno-histochemical analysis,we observed the expression of HTPAP in 377 cases,and evaluate the relationship between the protein expression and HTPAP genotype and haplotype.665 cases of HCC from Jan.2002 to June 2003 were enrolled for examining the SNPs by prosequencing 96,the association between postoperative DFS and HTPAP genotype and haplotype were investigated.
Results:By immuno-histochemical analysis in 377 cases of HCC,we observed the positive staining in 177 case(46.9%).Overally,two patterns of positive immunohistochemical staining have been occurred in cytoplasm.We observed intensified granular staining in cancer cells of the whole nodules,while the scattered positive staining of HCC in cancer nodules was also frequently present.Meanwhile, we found strong HTPAP staining in hyperplastic bile duct epithelial cell,but no positive staining in normal hepatic cells,liver cirrhotic cells,and infiltrating lymphocyte.Positive protein expression occurred more frequently in HCC cases with high metastasis potential compared with that with low potential(P=0.028).When we analysed the association of HTPAP genotypes and haplotypes with protein expression, we found the tag SNP +357(GG+GC) genotype and haplotypes(GCGGGT+GCGGAT) were negatively associated with HTPAP expression(P=0.002,P=0.001,respectively).No significance of HTPAP staining was found between tag SNP+1838(AG+GG) and AA genotype HCCs(P=0.130).
Kaplan-Meier analysis showed that the tag SNP+357 GG and GC was significantly associated with a decreased probability of postoperative DFS compared with +357CC genotype(P＜0.001),but we found no significance of postoperative DFS between the tagSNP+1838 GG,AG and AA genotype HCCs(P=0.282).We found the poorer postoperative DFS in HCCs of GCGGGT homozygotes and heterozygotes than that of others(P＜0.001),showing that GCGGGT homozygotes or heterozygotes was associated with a decreased probability of postoperative DFS compared with other haplotypes.
We recalculated the HRs for each tag SNP+357G/C genotyoes,and for haplotype of GCGGGT homozygotes,heterozygotes and others/others following adjustments for clinical characteristics.This analysis showed that only the +357 GG+GC genotype and GCGGGT homozygotes and heterozygotes were negatively associated with postoperative DFS(for+357GG genotype,P=0.021,HR(Hazard Ratios)=1.51, 95%C.I.=1.07-2.15,for+357GC genotype,P=0.030,HR=1.29,95%CI=1.03-1.62; for GCGGGT homozygotes,P=0.009,HR=1.59,95%C.I.=1.12-2.27,and for GCGGGT heterozygotes,P=0.022,HR=1.32,95%C.I.=1.04-1.67).
Conclusions:Our results show that variants at tumor suppressor have different effects on HCC metastasis potential.The tag SNP +357(GG+GC) genotype and haplotypes(GCGGGT+GCGGAT) were negatively associated with HTPAP expression. +357 GG+GC genotype and GCGGGT homozygote and heterozygotes of HTPAP are independently associated with a poor postoperative DFS in HCC patients after surgical resection,which indicates that +357 GG+GC genotype and GCGGGT homozygotes and heterozygotes might have great value in prediction of HCC metastasis potential(postoperative recurrence) and prognosis..
Conclusions
1.The tag SNP +357 GG+GC genotype and.haplotype G-C-G-G-G-T were significantly associated with higher metastatic potential risk in these Chinese HCC patients.
2.Different HTPAP haplotypes can influence the expression level,but none of them reach significance.The SNP genotype and haplotype in promoter could affect the transcriptional activity signficantly.
3.The different expression patterns might occurred between different HTPAP haplotype HCC associated with different HTPAP haplotype,and contributedly render changes in HCC phenotype such as invasion ability and metastasis potential indicating that the different HTPAP haplotypes could be the prognostic predicator of HCC metastasis potential.
4.Our results show that variants at tumor suppressor have different effects on HCC metastasis potential.+357GG+GC genotype and GCGGGT homozygote and heterozygotes are independently associated with a poor prognosis in HCC patients after surgical resection,which indicates they might have great value in prediction of HCC metastasis potential and prognosis
Potential application of this study
1.The tag SNP +357 GG+GC genotype and.haplotype G-C-G-G-G-T were significantly associated with higher metastatic potential risk,and might be a new biomarker of tumor metastasis.
2.Genotype +357 GG+GC and GCGGGT homozygote and heterozygotes are independently associated with a poor prognosis in HCC patients after surgical resection,which indicates they might have the potential of being a new genetic marker in prediction of metastasis and prognosis
Novelties
1.To the best of our knowledge,this is the first report investigating the association between the variants at HTPAP gene and HCC metastasis potential.
2.To the best of our knowledge,this is the first time to analyse different expression pattern between different HTPAP haplotype carriers in HCC by global gene-expression array to investigate the mechanism involved in the regulation of metastasis.
3.In addition,our data for the first time show that Genotype +357 GG+GC and GCGGGT homozygote and heterozygotes of HTPAP are independently associated with a decreased probability of postoperative DFS in HCC patients.
引文
1.Parkin DM,Bray F,Ferlay J,et al.Estimating the world cancer burden:Globocan 2000.Int J Cancer,2001,94:153-156
2.Parkin DM,Bray F,Ferlay J,et al.Global cancer statistics,2002.CA Cancer J Clin 2005,55:74-108.
3.Izzo F,Cremona F,Ruffolo F.Outcome of 67 patients with hepatocellular cancer detected during screening of 1125 patients with chronic hepatitis.Ann Surg 1998,227:513-518.
4.Zhou XD,Tang ZY,Yang BH.Experience of 1000 patients who underwent hepatectomy for small hepatocellular carcinoma.Cancer,2001,91:1479-1486.
5.Tang ZY,Yu YQ,Zhou XD.Hepatocellular carcinoma-Cause,treatment and metastasis.World J Gastroenterol,2001,7:445-454.
6.Li LD,Zhang SW,Lu FZ,et al.Research on characteristics of mortality spectrum and type composition of malignant tumors in China.Chin J Oncol,1997,19:323-328.
7.Yang BH,Zhang BH,Xu Y.Prospective study of early detection for primary liver cancer.J Cancer Res Clin Oncol,1997,123:357-360.
8.Tang ZY,Ye SL,Liu YK,et al.A decade's studies on metastasis of hepatocellular carcinoma. J Cancer Res Clin Oncol, 2004, 130: 187-196
9. Jemal A, Thomas A, Murray T, Thun M. Cancer statistics, 2002. CA Cancer J Clin, 2002, 52:23-47.
10. Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet, 2003,362:1907-1917.
11. Qin LX, Tang ZY, Sham JS I, et al. The association of chromosome 8p deletion and tumor metastasis in human hepatocellular carcinoma. Cancer Res,. 1999,59:5662-5
12. Qin LX, Tang ZY, Ye SL, et al. Chromosome 8p deletion is associated with metastasis of human hepatocellular carcinoma when high and low metastatic models are compared.. J Cancer Res Clin Oncol. 2001, 127:482-8.
13. Yang J, Qin LX, Ye SL, et al. The abnormalities of chromosome 8 in two hepatocellular carcinoma cell clones with the same genetic background and different metastatic potential J Cancer Res Clin Oncol. 2003 ,129:303-8..
14. Zhang LH, Qin LX, Ma ZC, et al. Allelic imbalance regions on chromosomes 8p, 17p and 19p related to metastasis of hepatocellular carcinoma: comparison between matched primary and metastatic lesions in 22 patients by genome-wide microsatellite analysis. J Cancer Res Clin Oncol, 2003 ,129:279-86.
15. Kanoh H, Kai M, Wada I, et al. Molecular characterization of the type 2 phosphatidic acid phosphatase.Chem Phys Lipids. 1999 ,98:119-26.
16. Ray ME, Yang ZQ, Albertson D, et al. Genomic and Expression Analysis of the 8pl 1—12 Amplicon in Human Breast Cancer Cell Lines. Cancer Res, 2004, 64, 40-47.
17. Yang ZQ, Albertson D, Ethier SP. Genomic organization of the 8p11～p12 amplicon in three breast cancer cell lines. Cancer Genetics and Cytogenetics, 2004, 155:57-62.
18. Garcia MJ, Pole JC, Chin SF, et al .A 1Mb minimal amplicon at 8p11-12 in breast cancer identifies new candidate oncogenes. Oncogene, 2005,24: 5235-5245.
19. Gelsi-Boyer, V, Orsetti B, Cervera N ,et al. Comprehensive Profiling of 8p11-12 Amplification in Breast Cancer. Mol Cancer Res ,2005,3:655-667.
20. Jenssen TK, Kuo W P, Stokke T. et al. Associations between gene expressions in breast cancer and patient survival. Hum Genet ,2002,111:411-420.
21. Wu X , Jia HL , Wang YF, et al. HTPAP gene on chromosome 8p is a candidate metastasis suppressorfor human hepatocellular carcinoma.Oncogene,2006,25:1832-1840.
22.Wang DG,Fan JB,Siao CJ,et al.Large—scale identification,mapping and genotyping of single nucleotide polymorphisms in the human genome.Science,1998,280:1077-1082.
23.杨昭庆,洪坤学,褚嘉佑.单核苷酸多态性的研究进展.国外医学遗传学分册,2000,23:4-8.
24.Wang Q,Rao S,Shen GQ,et al.Premature myocardial infarction novel suscep tibility locus on chromosome 1P34-36 identified by genomewide linkage analysis.Am J Hum Genet,2004;74:262
25.Yamada Y,Izawa H,Ichihara S,et al.Prediction of the risk of myocardial infarction from polymorphisms in candidate genes.N Engl J Med,2002;347:1916
26.Hall JM,LeeMK,Newman B,et al.Linkage of early-onset familial breast cancer to chromosome 17q21.Science,1990;250:1684
27.Horikawa Y,Oda N,CoxNJ,et al.Genetic variation in the gene encoding calpain -10 is associated with type 2 diabetes mellitus.Nat Genet,2000,26:163
28.ZhouW.Mapping genetic alterations in tumors with single nucleotide polymorphisms.CurrOp in Oncol,2003,15:50
29.Andreassen OA,Steen VM.Pharmacogenetics and tailored drug treatment in schizophrenia.TidsskrNor Laegeforen,2006;126:2400
30.Brumme ZL,Harrigan PR.The impact of human genetic variation on HIV disease in the ear of HAART.A IDS Rev,2006;8:78
31.Qin ZS,Niu T,Liu JS,et al.Partition-ligation-expectationmaximization algorithm for haplotype inference with singlenucleotide polymorphisms.Am J Hum Genet,2002,71:1242-1247.
32.Leabman MK,Huang CC,DeYoung J,et al.Natural variation in human embrane transporter genes reveals evolutionary and functional constraints.Proc Natl Acad Sci USA,2003,100:5896-5901.
33.Andrew G,Clark.The Role of Haplotypes in Candidate Gene Studies.Genetic Epidemiology,2004,27:321-333.
34.Johnson GC,Esposito L,Barratt BJ,et al.Haplotype tagging for the identification of common disease genes.Nat Genet,2001,29:233-323
35.Carlson CS,Eberle MA,Rieder MJ,et al.Additional SNPs and linkage- disequilibrium analyses are necessary for whole-genome association studies in humans. Nat Genet, 2003, 33:518-521.
1. Venter J C,AdamsM D,Myers E W, et al. The sequence of human genome. Science, 2001, 291:1304-1351.
2. Rubin G M. Comparing species . Nature, 2001,409: 820 - 821.
3. International Human genome Sequencing Consortium.Initial sequencing and analysis of the human genome. Nature, 2001, 409: 860 - 863.
4. The International SNP Map Working Group. A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature, 2001, 409: 860 - 863.
5. Sachidanandam R , Weissman D , Schmidt SC , et al . A map of human genome sequence variation containing 1. 42 million SNP. Nature , 2001 ,409 : 928-933.
6. Mullikin JC , Hunt SE , Cole CG, et al. An SNP map of human chromosome 22. Nature, 2000,407:516-520.
7. Eric L. Application of SNP technologies in medicine : lessons learned and future challenges. Genome Res , 2001 , 11:927-929.
8. Nyren P. Enzymatic method for continuous monitoring of DNA polymerase activity[J ]. Anal Biochem , 1987 ,167::235 -238.
9. Ronaghi M,Karamohamed S ,Pettersson B , et al . Real-time DNA sequencing using detection of PPi release . Anal Biochem ,1996 , 242:84 - 89.
10. Nyren P , Karamohamed S , Ronaghi M. Detection of single-base changes using a biolumino-metric primer extension assay .Anal Biochem , 1997 , 244:367 - 373.
11. Syvanen AC. Accessing genetic variation : genotyping single nucleotide polymorphisms [J ]. Nature Reviews Genetics , 2001 ,2 : 930 - 942.
12. Rafalski A. Application of single nucleotide polymorphisms in crop genetics. Current Opinion in Plant Biology , 2002 ,:94 -100.
13. Feng Q , Zhang Y,Hao P ,et al . Sequence and analysis of rice chromosome 4. Nature, 2002 ,420:316-320.
14. Nasu S , Suzuki J ,Ohta R ,et al . Search for and analysis of single nucleotide polymorphisms ( SNPs) in rice ( Oryza sativa , Oryzaruf ipogon) and establishment of SNP markers. DNA Research, 2002 ,9 :163 -171.
15. Li C , Zhang Y, Ying K,et al. Sequence variations of simple sequence repeats on chromosome24 in two subspecies of the Asian cultivated rice . Theor Appl Genet, 2004,108:392-400.
16. Yu JL , Hu S , Wong GKS , et al . A draft sequence of the rice genome ( Oryza sativa L. ssp. Indica). Science , 2002 ,296 :79 - 92.
17. Ronaghi M, Uhlen M, Nyren P. A sequencing method based on real-time pyrophosphate. Science , 1998 , 281 :363 - 365.
18. Zhou G, Li H, DeCamp D, et al. 2D differential in-gel electro-phoresis for the identification of esophageal scans cell cancer-specific p rotein markers. Mol Cell Proteomics, 2002, 1:117-124.
19. Craven RA, Totty N, Harnden P, et al. Laser cap ture microdissection and two-dimensional polyacrylamide gel electrophoresis: evaluation of tissue p reparation and samp le limitations. Am J Pathol, 2002, 160:815-822.
20. Bonner RF, Emmert2BuckM, Cole K, et al. Laser cap ture micro-dissection: molecular analysis of tissue. Science, 1997, 278:1481-1483.
21. Yu LR, Zeng R, Shao XX, et al. Identification of differentially exp ressed p roteins between human hepatoma and normal liver cell lines by two2dimensional electrophoresis and liquid chromatogram-physion trap mass spectrometry.Electrophoresis, 2000, 21:3058-3068.
22. Leiper J, NandiM, Torondel B, et al. Disrup tion ofmethylarginine metabolism impairs vascular homeostasis. Nat Med, 2007, 13:198-203.
23. International Union Against Cancer (UICC). Sobin LH, Wittekind C, eds. TNM classification of malignant tumors [M]. 6~(th) ed. New York: Wiley-Liss, 2002: 81-83
24. Gelsi-Boyer, V, Orsetti B, Cervera N ,et al. Comprehensive Profiling of 8p11-12 Amplification in Breast Cancer. Mol Cancer Res ,2005,3:655-667.
25. Wu X, Jia HL, Wang YF, et al. HTPAP gene on chromosome 8p is a candidate metastasis suppressor for human hepatocellular carcinoma. Oncogene, 2006, 25, 1832-1840.
26. Qin LX, Tang ZY, Sham JS I, et al. The association of chromosome 8p deletion and tumor metastasis in human hepatocellular carcinoma. Cancer Res,. 1999,59:5662-5
27. Pang JZ, Qin LX, Tang ZY,et al. Loss of heterozygosity at D8S298 is a predictor for long-term survival of patients with tumor-node-metastasis stage I of hepatocellular carcinoma. Clin Cancer Res. 2007,13,7363-7369.
28. Kubo M, Hata J , Ninomiya T, et al. A nonsynonymous SNP in PRKCH (protein kinase C Z) increases the risk of cerebral infarction .Nature genetics ,2007, 39:212-217
29. Sun T, Gao Y, Tan W, et al. A six-nucleotide insertion-deletion polymorphism in the CASP8 promoter is associated with susceptibility to multiple cancers. Nature genetics ,2007, 39:605-613c
30. Fonseca C ,Lindahl GE, Ponticos M, et al. A polymorphism in the CTGF promoter region associated with systemic sclerosis.N Eng J Med ,2007,35:1210-1220.
31. Sibley K, Rollinson S,Alexandra GA, et al. Functional FAS promoter polymorphism are associated with increased risk of acute myeloid leukemia. Cancer Res. 2003,63:4327-4330
32. Miyamoto Y, Mabuchi A, Shi DQ, et al.A functional polymorphism in the 5(?) UTR of GDF5 is associated with susceptibility to osteoarthritis . Nature genetics ,2007, 39:529-533.
33. Kong SY, Park JW , Lee JA, et al. Association between vascular endothelial growth factor gene polymorphism and survival in hepatocellular carcinoma patients. Hepatology, 2007,46:446-455.
34. Qin ZS, Niu T, Liu JS, et al. Partition-ligation-expectationmaximization algorithm for haplotype inference with singlenucleotide polymorphisms. Am J Hum Genet, 2002, 71:1242-1247.
35. Zhou J, Lu LW, Y MF, et al. Polymorphism of type I interferon receptor 1 promoter and their effects on chronic hepatitis B virus infection. Journal of hepatology 2007, 46:198-205.
1.Qin LX,Tang ZY,Sham JS I,et al.T he association of chromosome 8p deletion and tumor metastasis in human hepatocellular carcinoma.Cancer Res.1999,59:5662-5665
2.Qin LX,Tang ZY,Ye SL,et al.Chromosome 8p deletion is associated with metastasis of human hepatocellular carcinoma when high and low metastatic models are compared..J Cancer Res Clin Oncol.2001,127:482-488.
3.Yang J,Qin LX,Ye SL,et al.The abnormalities of chromosome 8 in two hepatocellular carcinoma cell clones with the same genetic background and different metastatic potential..J Cancer Res Clin Oncol.2003,129:303-308..
4.Zhang LH,Qin LX,Ma ZC,et al.Allelic imbalance regions on chromosomes 8p,17p and 19p related to metastasis of hepatocellular carcinoma:comparison between matched primary and metastatic lesions in 22 patients by genome-wide microsatellite analysis.J Cancer Res Clin Oncol.2003,129:279-286.
5.Kanoh H,Kai M,Wada I,et al.Molecular characterization of the type 2phosphatidic acid phosphatase.Chem Phys Lipids.1999,98:119-126.
6. Ray ME, Yang ZQ, Albertson D, et al. Genomic and Expression Analysis of the 8p11—12 Amplicon in Human Breast Cancer Cell Lines. Cancer Res, 2004, 64, 40-47.
7. Yang ZQ, Albertson D, Ethier SP. Genomic organization of the 8p11～p12 amplicon in three breast cancer cell lines. Cancer Genetics and Cytogenetics, 2004,155,57-62.
8. Garcia MJ, Pole JC, Chin SF, et al .A 1Mb minimal amplicon at 8p11—12 in breast cancer identifies new candidate oncogenes. Oncogene, 2005,24, 5235-5245.
9. Gelsi-Boyer, V, Orsetti B, Cervera N ,et al. Comprehensive Profiling of 8p11-12 Amplification in Breast Cancer. Mol Cancer Res ,2005,3:.655-667.
10. Wu X , Jia HL , Wang YF, et al. HTPAP gene on chromosome 8p is a candidate metastasis suppressorfor human hepatocellular carcinoma. Oncogene,2006, 25: 1832-1840.
11. Sun T, Gao Y, Tan W, et al. A six-nucleotide insertion-deletion polymorphism in the CASP8 promoter is associated with susceptibility to multiple cancers. Nature genetics ,2007, 39:605-613.
12. Fonseca C ,Lindahl GE, Ponticos M, et al. A polymorphism in the CTGF promoter region associated with systemic sclerosis. N Eng J Med,2007,35:1210-1220.
13. Sibley K, Rollinson S,Alexandra GA, et al. Functional FAS promoter polymorphism are associated with increased risk of acute myeloid leukemia. Cancer Res. 2003,63:4327-4330
14. Zhou J, Lu LW, Y MF, et al. Polymorphism of type I interferon receptor 1 promoter and their effects on chronic hepatitis B virus infection. Journal of hepatology 2007,46:198-205.
15. Miyamoto Y, Mabuchi A, Shi DQ, et al.A functional polymorphism in the 5(?) UTR of GDF5 is associated with susceptibility to osteoarthritis . Nature
16. Rosendahl J, Witt Heiko , Szmola R, et al.Chymotrypsin C (CTRC) variants that diminish activity or secretion are associated with chronic pancreatitis.Nat.genet, 2008:78-82.
17. Kubo M, Hata J, Ninomiyal T, et al.. A nonsynonymous SNP in PRKCH (proteinkinase C Z) increases the risk of cerebral infarction Nat.genet,2007:212-217.
18. Gregory SG , Schmidt S, Seth P,et al. Interleukin 7 receptor a chain (IL7R) shows allelic and functional association with multiple sclerosis. Nat.genet, 2007:83—91
19. Kathiresan S, Melander O, Guiducci Candace ? et al. Six new loci associated with blood low-density lipoprotein cholesterol, high-density lipoprotein cholesterol or riglycerides in humans. Nat.genet, ,2008: 189-97
20. Steinthorsdottir V, Thorleifsson G, Reynisdottir Inga , et al. A variant in CDKAL1 influences insulin response and risk of type 2 diabetes. Nat.genet,2007: 770-775.
21. Kwan T, Benovoy D, Diasl C, et al. Genome-wide analysis of transcript isoform variation in humans . Nat.genet, 2008:225-31.
22. Liu, S. , Altaian, R.B. Large scale study of protein domain distribution in the context of alternative splicing. Nucleic Acids Res. 2003,31: 4828-4835.
23. Lewis, B.P., Green, R.E. Brenner, S.E. Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans. Proc. Natl. Acad.Sci. USA ,2003,100:189-192.
24. Tian, B., Hu, J., Zhang, H. & Lutz, C.S. A large-scale analysis of mRNA polyadenylation of human and mouse genes. Nucleic Acids Res. 2005,33: 201-212.
25. Yan, J. & Marr, T.G. Computational analysis of 3'-ends of ESTs shows four classes of alternative polyadenylation in human, mouse, and rat. Genome Res. 2005,15:369-375
26. van Es MA, van Vught PW, Blauw HM, et al .Genetic variation in DPP6 is associated with susceptibility to amyotrophic lateral sclerosis. Nature genetics 2008,40:29-31
27. Parle-McDermott A, Pangilinan F,. Mills JL, et al .The 19-bp Deletion Polymorphism in Intron-1 of Dihydrofolate Reductase (DHFR) May Decrease Rather Than Increase Risk for Spina Bifida in the Irish Population. American Journal of Medical Genetics. 2007 ,143:1174-1180.
1.杨昭庆,洪坤学,褚嘉佑.单核苷酸多态性的研究进展[J].国外医学遗传学分册,2000,23:4-8.
2.Johnson GC,Esposito L,Barratt BJ,et al Haplotype tagging for the identification of common disease genes.Nat Genet,2001,29:233-237.
3.Leabman MK,Huang CC,DeYoung J,et al.Natural variation in human embrane transporter genes reveals evolutionary and functional constraints.Proc Natl Acad Sci USA,2003,100:5896-5901.
4.Andrew G,Clark.The Role of Haplotypes in Candidate Gene Studies.Genetic Epidemiology,2004,27:321-333.
5.[5]Carlson CS,Eberle MA,Rieder MJ,et al.Additional SNPs and linkage-disequilibrium analyses are necessary for whole-genome association studies in humans[J].Nat Genet,2003,33(4):518-521.
6.Ruano G,Kidd KK,Stephens JC,et al.Haplotype of multiple polymorphisms resolved by enzymatic amplification of single DNA molecules.Proc.Natl.Acad.Sci.U.S.A.,1998,87:6296-6300.
7.Michalatos BS,Tishkoff SA,Bentley KL,et al.Molecular haplotyping of genetic markers 10 kb apart by allele-specific long-range PCR.Nucleic Acids Res,1996,24:4841-4843.
8.Bradshaw MS,Bollekens JA,.Ruddle FH.A new vector for recombination-based cloning of large DNA fragments from yeast artificial chromosomes.Nucleic Acids Res.,1995,23:4850-4856.
9.Lizardi PM,Huang X,Zhu Z,et al.Mutation detection and single-molecule counting using isothermal rolling-circle amplification.Nat.Genet.,1998,19:225-232.
10.[10]Judson R,Stephen JC.Notes from the SNP vs.Haplotype front[J].Pharmacogenomics,2001,2(9):7-10.
11.Clark AG.Inference of haplotypes from PCR-amplified samples of diploid populations.Mol Biol Evol,1990,7:111-122.
12.Excoffier L,Slatkin M.Maximum-likelihood estimation of molecular Haplotype frequencies in a diploid population.Mol Biol Evol,1995,12:921-927.
13. Long JC, Williams RC, Urbanek M, et al. An E-M algorithm and testing strategy for multiple-locus haplotypes. Am J Hum Genet., 1995,56:799-810.
14. Fallin D , Schork NJ. Accuracy of haplotype frequency estimation for biallelic loci, via the expectation-maximization algorithm for unphased diopoid genotype data. Am J Hum Genet., 2000,67:947-959.
15. Thomas A. Accelerated Gene Counting for haplotype frequency estimation.Annals of Human Genetics, 2003,67:608-612.
16. Tregouet DA., Escolano S,.Tiret L, et al. A new algorithm for haplotype-based association analysis: the Stochastic-EM algorithm. Annals of Human Genetics. 2004,68:165-177.
17. Stephens M, Peter D. A Comparison of Bayesian Methods for Haplotype Reconstruction from Population Genotype Data. Am J Hum Genet, 2003,73:1162-1169.
18. Stephens M, Nicholas JS, Peter D, et al. A New Statistical Method for Haplotype Reconstruction from Population Data. Am J Hum Genet, 2001,68:978-989.
19. Niu T, Qin ZS, Xu X, Liu JS ,et al. Bayesian haplotype inference for multiple linked single-nucleotide polymorphisms. Am J Hum Genet ,2002,70:157-169.
20. Qin ZS, Niu T, Liu JS, et al. Partition-ligation-expectationmaximization algorithm for haplotype inference with singlenucleotide polymorphisms. Am J Hum Genet,2002, 71:1242-1247.
21. Nyholt DR. Genehunter: your "one-stop shop" for statistical genetic analysis. Hum Hered ,2002,53:2-7.
22. Sobel E, Lange K. Descent graphs in pedigree analysis: applications to haplotyping, location scores, and marker sharing statistics. Am J Hum Genet, 1996,58:1323-1337.
23. Abecasis GR, Cherny SS, Cookson WO, et al. MerlinFrapid analysis of dense genetic maps using sparse gene flow trees. Nat Genet ,2002,30:97-101.
24. Sabeti PC, Reich DE, Higgins JM, et al. Detecting recent positive selection in the human genome from haplotype structure. Nature,2002,419:832—837.
25. Zhou G, Zhai Y, Dong X, et al. Variants in TNFRSF5 locus and association analysis with hepatitis B virus (HBV) infection. Hum Mutat, 2004,23:99-100.
26. Pearce L, Hirschhorn N, Wu H, et al. Clarifying the PROGINS Allele Association in Ovarian and Breast Cancer Risk: A Haplotype-Based Analysis. Journal of the National Cancer Institute,2005,97:51-59.
27.陈汉奎,冯炳健,曾益新等。单体将家族性鼻咽癌易感基因定位于4P11-P14短区域。科学通报,2003,48:1776-1779.
28.Panguluri K,.Long LO,Chen W,et al.COX-2 gene promoter haplotypes and prostate cancer risk.Carcinogenesis,2004,25:961-966.
29.Peter K,Paul P,Chanock J,et al.Genetic Variation in the HSD17B1 Gene and Risk of Prostate Cancer.PLoS Genetics,2005,2:603-613.
30.Moon S,Holley S,Bodiwala D,et al.Associations Between G/A1229,A/G3944,T/C30875,C/T48200 and C/T65013 Genotypes and Haplotypes in the Vitamin D Receptor Gene,Ultraviolet Radiation and Susceptibility to Prostate.Annals of Human Genetics,2006,70:226-236.
31.David G C,Peter K,,Hankinson SE,et al.Haplotype analysis of common variants in the BRCA1 gene and risk of sporadic breast cancer.Breast Cancer Research,2004,17:171-175.
32.Feigelson S,David GC,Howard M,et al.Haplotype Analysis of the HSD17B1Gene and Risk of Breast Cancer:A Comprehensive Approach to Multicenter Analyses of Prospective Cohort Studies.Cancer Res,2006,66:2468-2475.
33.Benusiglio R,Lesueur F,Luccarini C,et al.Common variation in EMSY and risk of breast and ovarian cancer:a case-control study using HapMap tagging SNPs.BMC Cancer,2005,5:81-88.
34.Han W,Kang D,Lee E,et al.A Haplotype Analysis of HER-2 Gene Polymorphisms:Association with Breast Cancer Risk,HER-2 Protein Expression in theTumor,and Disease Recurrence in Korea.Clin Cancer Res,2005,11:4775-4782.
2.Parkin DM,Bray F,Ferlay J,et al.Global cancer statistics,2002.CA Cancer J Clin 2005,55:74-108.
3.Izzo F,Cremona F,Ruffolo F.Outcome of 67 patients with hepatocellular cancer detected during screening of 1125 patients with chronic hepatitis.Ann Surg 1998,227:513-518.
4.Zhou XD,Tang ZY,Yang BH.Experience of 1000 patients who underwent hepatectomy for small hepatocellular carcinoma.Cancer,2001,91:1479-1486.
5.Tang ZY,Yu YQ,Zhou XD.Hepatocellular carcinoma-Cause,treatment and metastasis.World J Gastroenterol,2001,7:445-454.
6.Li LD,Zhang SW,Lu FZ,et al.Research on characteristics of mortality spectrum and type composition of malignant tumors in China.Chin J Oncol,1997,19:323-328.
7.Yang BH,Zhang BH,Xu Y.Prospective study of early detection for primary liver cancer.J Cancer Res Clin Oncol,1997,123:357-360.
8.Tang ZY,Ye SL,Liu YK,et al.A decade's studies on metastasis of hepatocellular carcinoma. J Cancer Res Clin Oncol, 2004, 130: 187-196
9. Jemal A, Thomas A, Murray T, Thun M. Cancer statistics, 2002. CA Cancer J Clin, 2002, 52:23-47.
10. Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet, 2003,362:1907-1917.
11. Qin LX, Tang ZY, Sham JS I, et al. The association of chromosome 8p deletion and tumor metastasis in human hepatocellular carcinoma. Cancer Res,. 1999,59:5662-5
12. Qin LX, Tang ZY, Ye SL, et al. Chromosome 8p deletion is associated with metastasis of human hepatocellular carcinoma when high and low metastatic models are compared.. J Cancer Res Clin Oncol. 2001, 127:482-8.
13. Yang J, Qin LX, Ye SL, et al. The abnormalities of chromosome 8 in two hepatocellular carcinoma cell clones with the same genetic background and different metastatic potential J Cancer Res Clin Oncol. 2003 ,129:303-8..
14. Zhang LH, Qin LX, Ma ZC, et al. Allelic imbalance regions on chromosomes 8p, 17p and 19p related to metastasis of hepatocellular carcinoma: comparison between matched primary and metastatic lesions in 22 patients by genome-wide microsatellite analysis. J Cancer Res Clin Oncol, 2003 ,129:279-86.
15. Kanoh H, Kai M, Wada I, et al. Molecular characterization of the type 2 phosphatidic acid phosphatase.Chem Phys Lipids. 1999 ,98:119-26.
16. Ray ME, Yang ZQ, Albertson D, et al. Genomic and Expression Analysis of the 8pl 1—12 Amplicon in Human Breast Cancer Cell Lines. Cancer Res, 2004, 64, 40-47.
17. Yang ZQ, Albertson D, Ethier SP. Genomic organization of the 8p11～p12 amplicon in three breast cancer cell lines. Cancer Genetics and Cytogenetics, 2004, 155:57-62.
18. Garcia MJ, Pole JC, Chin SF, et al .A 1Mb minimal amplicon at 8p11-12 in breast cancer identifies new candidate oncogenes. Oncogene, 2005,24: 5235-5245.
19. Gelsi-Boyer, V, Orsetti B, Cervera N ,et al. Comprehensive Profiling of 8p11-12 Amplification in Breast Cancer. Mol Cancer Res ,2005,3:655-667.
20. Jenssen TK, Kuo W P, Stokke T. et al. Associations between gene expressions in breast cancer and patient survival. Hum Genet ,2002,111:411-420.
21. Wu X , Jia HL , Wang YF, et al. HTPAP gene on chromosome 8p is a candidate metastasis suppressorfor human hepatocellular carcinoma.Oncogene,2006,25:1832-1840.
22.Wang DG,Fan JB,Siao CJ,et al.Large—scale identification,mapping and genotyping of single nucleotide polymorphisms in the human genome.Science,1998,280:1077-1082.
23.杨昭庆,洪坤学,褚嘉佑.单核苷酸多态性的研究进展.国外医学遗传学分册,2000,23:4-8.
24.Wang Q,Rao S,Shen GQ,et al.Premature myocardial infarction novel suscep tibility locus on chromosome 1P34-36 identified by genomewide linkage analysis.Am J Hum Genet,2004;74:262
25.Yamada Y,Izawa H,Ichihara S,et al.Prediction of the risk of myocardial infarction from polymorphisms in candidate genes.N Engl J Med,2002;347:1916
26.Hall JM,LeeMK,Newman B,et al.Linkage of early-onset familial breast cancer to chromosome 17q21.Science,1990;250:1684
27.Horikawa Y,Oda N,CoxNJ,et al.Genetic variation in the gene encoding calpain -10 is associated with type 2 diabetes mellitus.Nat Genet,2000,26:163
28.ZhouW.Mapping genetic alterations in tumors with single nucleotide polymorphisms.CurrOp in Oncol,2003,15:50
29.Andreassen OA,Steen VM.Pharmacogenetics and tailored drug treatment in schizophrenia.TidsskrNor Laegeforen,2006;126:2400
30.Brumme ZL,Harrigan PR.The impact of human genetic variation on HIV disease in the ear of HAART.A IDS Rev,2006;8:78
31.Qin ZS,Niu T,Liu JS,et al.Partition-ligation-expectationmaximization algorithm for haplotype inference with singlenucleotide polymorphisms.Am J Hum Genet,2002,71:1242-1247.
32.Leabman MK,Huang CC,DeYoung J,et al.Natural variation in human embrane transporter genes reveals evolutionary and functional constraints.Proc Natl Acad Sci USA,2003,100:5896-5901.
33.Andrew G,Clark.The Role of Haplotypes in Candidate Gene Studies.Genetic Epidemiology,2004,27:321-333.
34.Johnson GC,Esposito L,Barratt BJ,et al.Haplotype tagging for the identification of common disease genes.Nat Genet,2001,29:233-323
35.Carlson CS,Eberle MA,Rieder MJ,et al.Additional SNPs and linkage- disequilibrium analyses are necessary for whole-genome association studies in humans. Nat Genet, 2003, 33:518-521.
1. Venter J C,AdamsM D,Myers E W, et al. The sequence of human genome. Science, 2001, 291:1304-1351.
2. Rubin G M. Comparing species . Nature, 2001,409: 820 - 821.
3. International Human genome Sequencing Consortium.Initial sequencing and analysis of the human genome. Nature, 2001, 409: 860 - 863.
4. The International SNP Map Working Group. A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature, 2001, 409: 860 - 863.
5. Sachidanandam R , Weissman D , Schmidt SC , et al . A map of human genome sequence variation containing 1. 42 million SNP. Nature , 2001 ,409 : 928-933.
6. Mullikin JC , Hunt SE , Cole CG, et al. An SNP map of human chromosome 22. Nature, 2000,407:516-520.
7. Eric L. Application of SNP technologies in medicine : lessons learned and future challenges. Genome Res , 2001 , 11:927-929.
8. Nyren P. Enzymatic method for continuous monitoring of DNA polymerase activity[J ]. Anal Biochem , 1987 ,167::235 -238.
9. Ronaghi M,Karamohamed S ,Pettersson B , et al . Real-time DNA sequencing using detection of PPi release . Anal Biochem ,1996 , 242:84 - 89.
10. Nyren P , Karamohamed S , Ronaghi M. Detection of single-base changes using a biolumino-metric primer extension assay .Anal Biochem , 1997 , 244:367 - 373.
11. Syvanen AC. Accessing genetic variation : genotyping single nucleotide polymorphisms [J ]. Nature Reviews Genetics , 2001 ,2 : 930 - 942.
12. Rafalski A. Application of single nucleotide polymorphisms in crop genetics. Current Opinion in Plant Biology , 2002 ,:94 -100.
13. Feng Q , Zhang Y,Hao P ,et al . Sequence and analysis of rice chromosome 4. Nature, 2002 ,420:316-320.
14. Nasu S , Suzuki J ,Ohta R ,et al . Search for and analysis of single nucleotide polymorphisms ( SNPs) in rice ( Oryza sativa , Oryzaruf ipogon) and establishment of SNP markers. DNA Research, 2002 ,9 :163 -171.
15. Li C , Zhang Y, Ying K,et al. Sequence variations of simple sequence repeats on chromosome24 in two subspecies of the Asian cultivated rice . Theor Appl Genet, 2004,108:392-400.
16. Yu JL , Hu S , Wong GKS , et al . A draft sequence of the rice genome ( Oryza sativa L. ssp. Indica). Science , 2002 ,296 :79 - 92.
17. Ronaghi M, Uhlen M, Nyren P. A sequencing method based on real-time pyrophosphate. Science , 1998 , 281 :363 - 365.
18. Zhou G, Li H, DeCamp D, et al. 2D differential in-gel electro-phoresis for the identification of esophageal scans cell cancer-specific p rotein markers. Mol Cell Proteomics, 2002, 1:117-124.
19. Craven RA, Totty N, Harnden P, et al. Laser cap ture microdissection and two-dimensional polyacrylamide gel electrophoresis: evaluation of tissue p reparation and samp le limitations. Am J Pathol, 2002, 160:815-822.
20. Bonner RF, Emmert2BuckM, Cole K, et al. Laser cap ture micro-dissection: molecular analysis of tissue. Science, 1997, 278:1481-1483.
21. Yu LR, Zeng R, Shao XX, et al. Identification of differentially exp ressed p roteins between human hepatoma and normal liver cell lines by two2dimensional electrophoresis and liquid chromatogram-physion trap mass spectrometry.Electrophoresis, 2000, 21:3058-3068.
22. Leiper J, NandiM, Torondel B, et al. Disrup tion ofmethylarginine metabolism impairs vascular homeostasis. Nat Med, 2007, 13:198-203.
23. International Union Against Cancer (UICC). Sobin LH, Wittekind C, eds. TNM classification of malignant tumors [M]. 6~(th) ed. New York: Wiley-Liss, 2002: 81-83
24. Gelsi-Boyer, V, Orsetti B, Cervera N ,et al. Comprehensive Profiling of 8p11-12 Amplification in Breast Cancer. Mol Cancer Res ,2005,3:655-667.
25. Wu X, Jia HL, Wang YF, et al. HTPAP gene on chromosome 8p is a candidate metastasis suppressor for human hepatocellular carcinoma. Oncogene, 2006, 25, 1832-1840.
26. Qin LX, Tang ZY, Sham JS I, et al. The association of chromosome 8p deletion and tumor metastasis in human hepatocellular carcinoma. Cancer Res,. 1999,59:5662-5
27. Pang JZ, Qin LX, Tang ZY,et al. Loss of heterozygosity at D8S298 is a predictor for long-term survival of patients with tumor-node-metastasis stage I of hepatocellular carcinoma. Clin Cancer Res. 2007,13,7363-7369.
28. Kubo M, Hata J , Ninomiya T, et al. A nonsynonymous SNP in PRKCH (protein kinase C Z) increases the risk of cerebral infarction .Nature genetics ,2007, 39:212-217
29. Sun T, Gao Y, Tan W, et al. A six-nucleotide insertion-deletion polymorphism in the CASP8 promoter is associated with susceptibility to multiple cancers. Nature genetics ,2007, 39:605-613c
30. Fonseca C ,Lindahl GE, Ponticos M, et al. A polymorphism in the CTGF promoter region associated with systemic sclerosis.N Eng J Med ,2007,35:1210-1220.
31. Sibley K, Rollinson S,Alexandra GA, et al. Functional FAS promoter polymorphism are associated with increased risk of acute myeloid leukemia. Cancer Res. 2003,63:4327-4330
32. Miyamoto Y, Mabuchi A, Shi DQ, et al.A functional polymorphism in the 5(?) UTR of GDF5 is associated with susceptibility to osteoarthritis . Nature genetics ,2007, 39:529-533.
33. Kong SY, Park JW , Lee JA, et al. Association between vascular endothelial growth factor gene polymorphism and survival in hepatocellular carcinoma patients. Hepatology, 2007,46:446-455.
34. Qin ZS, Niu T, Liu JS, et al. Partition-ligation-expectationmaximization algorithm for haplotype inference with singlenucleotide polymorphisms. Am J Hum Genet, 2002, 71:1242-1247.
35. Zhou J, Lu LW, Y MF, et al. Polymorphism of type I interferon receptor 1 promoter and their effects on chronic hepatitis B virus infection. Journal of hepatology 2007, 46:198-205.
1.Qin LX,Tang ZY,Sham JS I,et al.T he association of chromosome 8p deletion and tumor metastasis in human hepatocellular carcinoma.Cancer Res.1999,59:5662-5665
2.Qin LX,Tang ZY,Ye SL,et al.Chromosome 8p deletion is associated with metastasis of human hepatocellular carcinoma when high and low metastatic models are compared..J Cancer Res Clin Oncol.2001,127:482-488.
3.Yang J,Qin LX,Ye SL,et al.The abnormalities of chromosome 8 in two hepatocellular carcinoma cell clones with the same genetic background and different metastatic potential..J Cancer Res Clin Oncol.2003,129:303-308..
4.Zhang LH,Qin LX,Ma ZC,et al.Allelic imbalance regions on chromosomes 8p,17p and 19p related to metastasis of hepatocellular carcinoma:comparison between matched primary and metastatic lesions in 22 patients by genome-wide microsatellite analysis.J Cancer Res Clin Oncol.2003,129:279-286.
5.Kanoh H,Kai M,Wada I,et al.Molecular characterization of the type 2phosphatidic acid phosphatase.Chem Phys Lipids.1999,98:119-126.
6. Ray ME, Yang ZQ, Albertson D, et al. Genomic and Expression Analysis of the 8p11—12 Amplicon in Human Breast Cancer Cell Lines. Cancer Res, 2004, 64, 40-47.
7. Yang ZQ, Albertson D, Ethier SP. Genomic organization of the 8p11～p12 amplicon in three breast cancer cell lines. Cancer Genetics and Cytogenetics, 2004,155,57-62.
8. Garcia MJ, Pole JC, Chin SF, et al .A 1Mb minimal amplicon at 8p11—12 in breast cancer identifies new candidate oncogenes. Oncogene, 2005,24, 5235-5245.
9. Gelsi-Boyer, V, Orsetti B, Cervera N ,et al. Comprehensive Profiling of 8p11-12 Amplification in Breast Cancer. Mol Cancer Res ,2005,3:.655-667.
10. Wu X , Jia HL , Wang YF, et al. HTPAP gene on chromosome 8p is a candidate metastasis suppressorfor human hepatocellular carcinoma. Oncogene,2006, 25: 1832-1840.
11. Sun T, Gao Y, Tan W, et al. A six-nucleotide insertion-deletion polymorphism in the CASP8 promoter is associated with susceptibility to multiple cancers. Nature genetics ,2007, 39:605-613.
12. Fonseca C ,Lindahl GE, Ponticos M, et al. A polymorphism in the CTGF promoter region associated with systemic sclerosis. N Eng J Med,2007,35:1210-1220.
13. Sibley K, Rollinson S,Alexandra GA, et al. Functional FAS promoter polymorphism are associated with increased risk of acute myeloid leukemia. Cancer Res. 2003,63:4327-4330
14. Zhou J, Lu LW, Y MF, et al. Polymorphism of type I interferon receptor 1 promoter and their effects on chronic hepatitis B virus infection. Journal of hepatology 2007,46:198-205.
15. Miyamoto Y, Mabuchi A, Shi DQ, et al.A functional polymorphism in the 5(?) UTR of GDF5 is associated with susceptibility to osteoarthritis . Nature
16. Rosendahl J, Witt Heiko , Szmola R, et al.Chymotrypsin C (CTRC) variants that diminish activity or secretion are associated with chronic pancreatitis.Nat.genet, 2008:78-82.
17. Kubo M, Hata J, Ninomiyal T, et al.. A nonsynonymous SNP in PRKCH (proteinkinase C Z) increases the risk of cerebral infarction Nat.genet,2007:212-217.
18. Gregory SG , Schmidt S, Seth P,et al. Interleukin 7 receptor a chain (IL7R) shows allelic and functional association with multiple sclerosis. Nat.genet, 2007:83—91
19. Kathiresan S, Melander O, Guiducci Candace ? et al. Six new loci associated with blood low-density lipoprotein cholesterol, high-density lipoprotein cholesterol or riglycerides in humans. Nat.genet, ,2008: 189-97
20. Steinthorsdottir V, Thorleifsson G, Reynisdottir Inga , et al. A variant in CDKAL1 influences insulin response and risk of type 2 diabetes. Nat.genet,2007: 770-775.
21. Kwan T, Benovoy D, Diasl C, et al. Genome-wide analysis of transcript isoform variation in humans . Nat.genet, 2008:225-31.
22. Liu, S. , Altaian, R.B. Large scale study of protein domain distribution in the context of alternative splicing. Nucleic Acids Res. 2003,31: 4828-4835.
23. Lewis, B.P., Green, R.E. Brenner, S.E. Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans. Proc. Natl. Acad.Sci. USA ,2003,100:189-192.
24. Tian, B., Hu, J., Zhang, H. & Lutz, C.S. A large-scale analysis of mRNA polyadenylation of human and mouse genes. Nucleic Acids Res. 2005,33: 201-212.
25. Yan, J. & Marr, T.G. Computational analysis of 3'-ends of ESTs shows four classes of alternative polyadenylation in human, mouse, and rat. Genome Res. 2005,15:369-375
26. van Es MA, van Vught PW, Blauw HM, et al .Genetic variation in DPP6 is associated with susceptibility to amyotrophic lateral sclerosis. Nature genetics 2008,40:29-31
27. Parle-McDermott A, Pangilinan F,. Mills JL, et al .The 19-bp Deletion Polymorphism in Intron-1 of Dihydrofolate Reductase (DHFR) May Decrease Rather Than Increase Risk for Spina Bifida in the Irish Population. American Journal of Medical Genetics. 2007 ,143:1174-1180.
1.杨昭庆,洪坤学,褚嘉佑.单核苷酸多态性的研究进展[J].国外医学遗传学分册,2000,23:4-8.
2.Johnson GC,Esposito L,Barratt BJ,et al Haplotype tagging for the identification of common disease genes.Nat Genet,2001,29:233-237.
3.Leabman MK,Huang CC,DeYoung J,et al.Natural variation in human embrane transporter genes reveals evolutionary and functional constraints.Proc Natl Acad Sci USA,2003,100:5896-5901.
4.Andrew G,Clark.The Role of Haplotypes in Candidate Gene Studies.Genetic Epidemiology,2004,27:321-333.
5.[5]Carlson CS,Eberle MA,Rieder MJ,et al.Additional SNPs and linkage-disequilibrium analyses are necessary for whole-genome association studies in humans[J].Nat Genet,2003,33(4):518-521.
6.Ruano G,Kidd KK,Stephens JC,et al.Haplotype of multiple polymorphisms resolved by enzymatic amplification of single DNA molecules.Proc.Natl.Acad.Sci.U.S.A.,1998,87:6296-6300.
7.Michalatos BS,Tishkoff SA,Bentley KL,et al.Molecular haplotyping of genetic markers 10 kb apart by allele-specific long-range PCR.Nucleic Acids Res,1996,24:4841-4843.
8.Bradshaw MS,Bollekens JA,.Ruddle FH.A new vector for recombination-based cloning of large DNA fragments from yeast artificial chromosomes.Nucleic Acids Res.,1995,23:4850-4856.
9.Lizardi PM,Huang X,Zhu Z,et al.Mutation detection and single-molecule counting using isothermal rolling-circle amplification.Nat.Genet.,1998,19:225-232.
10.[10]Judson R,Stephen JC.Notes from the SNP vs.Haplotype front[J].Pharmacogenomics,2001,2(9):7-10.
11.Clark AG.Inference of haplotypes from PCR-amplified samples of diploid populations.Mol Biol Evol,1990,7:111-122.
12.Excoffier L,Slatkin M.Maximum-likelihood estimation of molecular Haplotype frequencies in a diploid population.Mol Biol Evol,1995,12:921-927.
13. Long JC, Williams RC, Urbanek M, et al. An E-M algorithm and testing strategy for multiple-locus haplotypes. Am J Hum Genet., 1995,56:799-810.
14. Fallin D , Schork NJ. Accuracy of haplotype frequency estimation for biallelic loci, via the expectation-maximization algorithm for unphased diopoid genotype data. Am J Hum Genet., 2000,67:947-959.
15. Thomas A. Accelerated Gene Counting for haplotype frequency estimation.Annals of Human Genetics, 2003,67:608-612.
16. Tregouet DA., Escolano S,.Tiret L, et al. A new algorithm for haplotype-based association analysis: the Stochastic-EM algorithm. Annals of Human Genetics. 2004,68:165-177.
17. Stephens M, Peter D. A Comparison of Bayesian Methods for Haplotype Reconstruction from Population Genotype Data. Am J Hum Genet, 2003,73:1162-1169.
18. Stephens M, Nicholas JS, Peter D, et al. A New Statistical Method for Haplotype Reconstruction from Population Data. Am J Hum Genet, 2001,68:978-989.
19. Niu T, Qin ZS, Xu X, Liu JS ,et al. Bayesian haplotype inference for multiple linked single-nucleotide polymorphisms. Am J Hum Genet ,2002,70:157-169.
20. Qin ZS, Niu T, Liu JS, et al. Partition-ligation-expectationmaximization algorithm for haplotype inference with singlenucleotide polymorphisms. Am J Hum Genet,2002, 71:1242-1247.
21. Nyholt DR. Genehunter: your "one-stop shop" for statistical genetic analysis. Hum Hered ,2002,53:2-7.
22. Sobel E, Lange K. Descent graphs in pedigree analysis: applications to haplotyping, location scores, and marker sharing statistics. Am J Hum Genet, 1996,58:1323-1337.
23. Abecasis GR, Cherny SS, Cookson WO, et al. MerlinFrapid analysis of dense genetic maps using sparse gene flow trees. Nat Genet ,2002,30:97-101.
24. Sabeti PC, Reich DE, Higgins JM, et al. Detecting recent positive selection in the human genome from haplotype structure. Nature,2002,419:832—837.
25. Zhou G, Zhai Y, Dong X, et al. Variants in TNFRSF5 locus and association analysis with hepatitis B virus (HBV) infection. Hum Mutat, 2004,23:99-100.
26. Pearce L, Hirschhorn N, Wu H, et al. Clarifying the PROGINS Allele Association in Ovarian and Breast Cancer Risk: A Haplotype-Based Analysis. Journal of the National Cancer Institute,2005,97:51-59.
27.陈汉奎,冯炳健,曾益新等。单体将家族性鼻咽癌易感基因定位于4P11-P14短区域。科学通报,2003,48:1776-1779.
28.Panguluri K,.Long LO,Chen W,et al.COX-2 gene promoter haplotypes and prostate cancer risk.Carcinogenesis,2004,25:961-966.
29.Peter K,Paul P,Chanock J,et al.Genetic Variation in the HSD17B1 Gene and Risk of Prostate Cancer.PLoS Genetics,2005,2:603-613.
30.Moon S,Holley S,Bodiwala D,et al.Associations Between G/A1229,A/G3944,T/C30875,C/T48200 and C/T65013 Genotypes and Haplotypes in the Vitamin D Receptor Gene,Ultraviolet Radiation and Susceptibility to Prostate.Annals of Human Genetics,2006,70:226-236.
31.David G C,Peter K,,Hankinson SE,et al.Haplotype analysis of common variants in the BRCA1 gene and risk of sporadic breast cancer.Breast Cancer Research,2004,17:171-175.
32.Feigelson S,David GC,Howard M,et al.Haplotype Analysis of the HSD17B1Gene and Risk of Breast Cancer:A Comprehensive Approach to Multicenter Analyses of Prospective Cohort Studies.Cancer Res,2006,66:2468-2475.
33.Benusiglio R,Lesueur F,Luccarini C,et al.Common variation in EMSY and risk of breast and ovarian cancer:a case-control study using HapMap tagging SNPs.BMC Cancer,2005,5:81-88.
34.Han W,Kang D,Lee E,et al.A Haplotype Analysis of HER-2 Gene Polymorphisms:Association with Breast Cancer Risk,HER-2 Protein Expression in theTumor,and Disease Recurrence in Korea.Clin Cancer Res,2005,11:4775-4782.