Genetic Susceptibility to Loss of Tumor Suppressor Gene

癌症是一种复杂的疾病，与遗传性和获得性遗传和/或表观遗传变化有关，这些变化随着时间的推移而发生，可能是由环境和内源性暴露引起的。癌症的关键特征包括肿瘤抑制基因功能的丧失和/或原癌基因对癌基因的激活。这些变化与基因表达的改变有关，这些基因表达的改变导致控制快速生长的体细胞或干细胞克隆的细胞周期、进展和增殖的相关信号通路功能障碍。最终，这种基因剂量的不平衡会导致基因组的不稳定，导致癌前细胞的克隆性衍生，导致与癌症发展相关的不受控制的程序性细胞死亡和增殖。这些特征是常见的，并且已经在人类和近亲繁殖的实验室啮齿动物中作为替代品进行了广泛的研究。我们观察到B6.129/6-Trp53tm1Brd和B6C3F1-Trp53tm1BrdN12(回交第12代)单倍体缺陷小鼠在暴露于人类致癌物(包括电离辐射、苯、环磷酰胺、马法兰等)后发生造血干细胞(HSC)肿瘤。与B6.129-Trp53tm1Brd N5或N12野生型小鼠相比，B6.129-Trp53tm1Brd N5或N12野生型小鼠(C3H、129或DBA/2品系)非常迅速(！O2-3倍快)，发病率(60%-100%)更高。DBA/2等位基因改变了这种对电离辐射的反应。这些致癌物诱导的HSC肿瘤具有更高的发生率和程度，涉及11号染色体上Trp53基因座的杂合性丢失和全基因组，通过使用菌株特异性微卫星(SSLP)标记和阵列比较基因组杂交(ACGH)来确定可重现的染色体特异的得失模式。在电离辐射诱导的肿瘤中，致癌物特异性LOH的诱导模式与非连接(未能维持有丝分裂纺锤体和检查点)和体细胞有丝分裂重组(错误的同源或非同源序列定向修复)是一致的。 我们研究了错误分离(非错配导致数字染色体丢失)和/或非同源和同源及非同源序列定向修复的作用机制。利用每个基因在每个座位上的两种不同的等位基因形式，我们已经确定杂合性的丧失与P53抑癌基因及其功能的丧失有关。此外，P53单倍体不足会加剧基因组的不稳定性，从而导致基因拷贝数的单倍型特异性改变。我们利用菌株SSLP标记、单核苷酸多态(SNP)标记和位点特异性基因缺失(ACGH)标记，在C3B6F1-Trpp53单倍体缺陷小鼠淋巴瘤中定位了电离辐射诱导的等位基因特异性杂合性缺失(LOH)位点，并确定了可能的肿瘤抑制基因的位点。此外，我们已经开始了对杂合性缺失表型遗传易感性的潜在研究(通过菌株在DNA损伤和修复方面的特定差异)，以确定数量性状基因座(QTL)，从而利用单倍型-表型关联研究识别与之相关的高渗透数量基因和低外显基因。目前正在利用多种方法。其中包括利用B6xD2杂交的F1和F2杂交、BxD重组自交系和单倍型多样性菌株(16个菌株由PerLegen和NIEHS进行密集的基因分型)进行表型鉴定和基因鉴定，以鉴定与所开发的定量DNA修复表型分离的单倍型。在相关研究中，我们已经表明，DNA氧化(细胞内氧化条件)加剧了电离辐射暴露和淋巴瘤发生后Trp53野生型等位基因杂合性的丧失。通过体外造血干细胞培养克隆分析肿瘤中依赖菌株的LOH，将使我们能够在基因组水平上研究抑癌基因单倍体不足在LOH中的作用，并识别改变LOH表型易感性的基因。 这种方法的威力是基于个体的遗传差异，以及利用连锁不平衡分析和单倍型关联研究在该表型的表现中差异表达的基因和表型之间的统计关联。候选基因的推断和鉴定也可以基于与DNA损伤和修复、细胞凋亡和细胞增殖有关的信号和功能通路的知识，使用依赖于菌株的单倍型关联。我们可以进一步扩展这一方法，使用原代培养的造血干细胞(HSC)进行体外比较研究，并在两个物种中进行单核苷酸多态(SNP；单倍型)关联研究，通过将特定的基因类型(单倍型)与基于早期DNA损伤和修复途径对电离辐射的反应以及生物学表型(细胞凋亡、DSB修复生物标志物、DNA修复分析等)的基因表达表型相关联来确定候选基因的共同点。

Cancer is a complex disease associated with both inherited and acquired genetic and/or epigenetic changes that occur over time that may arise from both environmental and endogenous exposures. Critical features of cancer include the loss of tumor suppressor gene function and/or activation of proto-oncogenes to oncogenes. These changes are associated with alterations in gene expression that lead to the dysfunction of associated signaling pathways that control the cell cycle, progression, and proliferation of clones of rapidly growing somatic or stem cells. Ultimately, this imbalance in gene dosage leads to genomic instability that results in clonal derivation of pre-cancerous cells with uncontrolled programmed cell death and proliferation associated with the development of cancer. These features are common and have been extensively studied in both humans and inbred laboratory rodents as surrogates. We have observed that B6.129/6-Trp53tm1Brd and B6C3F1-Trp53tm1Brd N12 (12th backcross generation) haploinsufficient mice develop hematopoietic stem cell (HSC) neoplasms after exposure to human carcinogens (including ionizing radiation, benzene, cyclophosphamide, melphalan, etc.) very rapidly (!O2-3x faster) and with greater prevalence (60-100%) than either B6.129-Trp53tm1Brd N5 or N12 wild type mice compared to F1 intercross mice (C3H, 129, or DBA/2 strains). DBA/2 alleles modify this response to ionizing radiation. These carcinogen-induced HSC neoplasms have an increased prevalence and magnitude for the loss of heterozygosity involving the Trp53 locus on chromosome 11 and genomic wide with reproducible chromosome specific patterns of gains or losses as determined by using both strain specific microsattelite (SSLP) markers and array comparative genomic hybridization (aCGH). The carcinogen specific induced pattern of LOH in ionizing radiation induced tumors is consistent with both non-dysjunction (failure to maintain the mitotic spindle apparatus and checkpoint) and somatic cell mitotic recombination (errors in homologous or non-homologous sequence directed repair). We have investigated the mechanism for the role of mis-segregation (non-dysjunction resulting in numerical chromosome loss) and/or non-homologous and homologous and non-homologous sequence directed repair. Using with two different allelic forms of each gene at each locus, we have established that loss of heterozygosity is associated with the loss of the p53 tumor suppressor gene and its function. Furthermore, genomic instability is exacerbated by p53 haploinsufficiency resulting in reproducible haplotype specific changes in gene copy number. We have mapped sites of ionizing radiation induced allele specific LOH and identify sites of putative tumor suppressor genes in lymphomas from C3B6F1-Trpp53 haploinsufficient mice using strain-SSLP markers, single nucleotide polymorphic (SNP) markers, and loss of locus specific genes (aCGH). In addition, we have initiated investigation of the potential for genetic susceptibility to the loss of heterozygosity phenotype (through strain specific differences in DNA damage and repair) in order to identify quantitative trait loci (QTL) leading to identification of both the associated highly penetrant quantitative genes that confer susceptibility or resistance and the lower penetrance genes that modify susceptibility using haplotype-phenotype association studies. Multiple approaches are being utilized. These include the use of F1 and F2 intercrosses of B6xD2 intercrosses, BxD recombinant inbred lines, and haplotype diverse strains (16 strains densely genotyped by Perlegen and NIEHS) for phenotyping and genotyping for identification of haplotypes that segregate with the quantitative DNA repair phenotype developed. In related studies, we have shown that DNA oxidation (intracellular pro-oxidant conditions) exacerbates loss of heterozygosity of the Trp53 wild type allele following ionizing radiation exposure and lymphoma development. The quantification of strain dependent LOH in tumors by clonal analysis using hematopoietic stem cell culture in vitro will allow us to investigate the role of tumor suppressor gene haploinsufficiency at the genomic level on LOH and identify genes that modify the susceptibility to LOH phenotype. The power of this approach is based on individual genetic differences and the statistical association between the phenotype and the genes that are differentially expressed in the manifestation of this phenotype using linkage disequilibrium analysis and haplotype association studies. Deduction and identification of candidate genes can also be based on knowledge of the signaling and functional pathways involved in DNA damage and repair, apoptosis, and cellular proliferation using strain dependent haplotype association. We can extend this approach further by conducting comparative mouse and human studies in vitro using primary culture of hematopoietic stem cells (HSC) and conducting single nucleotide polymorphism (SNP; haplotype) association studies in both species to identify candidate genes in common by correlating specific genotypes (haplotypes) with gene expression phenotypes based on the early DNA damage and repair pathway response to ionizing radiation and biological phenotypes (apoptosis, DSB repair biomarkers, DNA repair assays, etc).