Identification of Single Nucleotide Polymorphisms in Cancer-Related Genes

个体的遗传背景在他们对癌症的易感性、疾病进展和对治疗的反应中起着重要作用。遗传变异可以作为理解复杂疾病的标记，包括癌症、免疫/炎症反应和艾滋病。人类和其他基因组序列的出现以及键入大量遗传标记的能力导致了对大大增强的计算和分析方法的需求。与纪念斯隆凯特琳癌症研究所的科学家合作，我们完成了一项关于有家族病史的德系犹太人乳腺癌全基因组关联研究。扫描确定了几个具有潜在显著关联的位点。从这些数据中，我们选择了一个基因座用于重复队列的随访研究。我们能够确认FGFR2基因与几个欧美人群的乳腺癌有关，也与德系犹太人的癌症有关。此外，一个包含两个基因RNF146和ECHDC1的位点被确定与德系犹太人的乳腺癌呈正相关。RNF146与调节雌激素受体的蛋白具有共同的基序。在雌激素受体功能检测中，RNF146抑制雌激素反应。为了进一步表征该蛋白，我们在细菌细胞中表达了该产物。我们正在与David Waugh合作，结晶蛋白质以确定其三维结构。此外，我们正在与Allan Weissman合作评估预测的泛素e3样连接酶活性，该蛋白预计将拥有。补体基因与年龄相关性黄斑变性通过研究补体B和C2 （CFH, C2）基因的变异，我们已经确定了该基因与年龄相关性黄斑变性（AMD）患者的关联。老年性黄斑变性是导致老年人失明的主要原因，据估计有多达1000万美国人受其影响。通过检测BF和C2基因的遗传变异，鉴定出具有保护作用的单倍型。补体途径在对致癌病原体的反应中是重要的。对先天免疫反应的这一重要组成部分的进一步研究可能会导致对人类疾病的深入了解。去除CFH附近的CFHR1基因的缺失对AMD具有高度的保护作用。我们已经从年龄相关性眼病研究的受试者中获得了3000多个dna，并正在对它们进行分型以确定相关标记，以了解关联的细节并确定其他位点。最近发表的数据表明AMD与toll样受体3 （TLR3）基因有关。然而，我们在AREDS队列中输入了这种变异，并与来自其他7个中心的数据一起未能复制这种关联。HH/PTCH通路已被证明在几乎所有基底细胞癌和部分髓母细胞瘤中发生突变。此外，许多肿瘤显示HH/PTCH通路的配体依赖性激活，包括胰腺、前列腺、胃肠道和小细胞肺肿瘤。类固醇样的天然产物分子环巴胺特异性地与SMO结合，SMO是HH/PTCH通路的下游调节因子。环巴胺已被证明可以抑制HH/ ptch激活的细胞系和异种移植物的生长。为了证明环巴胺是否抑制HH/PTCH靶基因的表达，我们对前列腺（DU-145, LnCaP），胃（AGS）和乳腺癌（SK-BR3）细胞系进行了处理，并证实环巴胺抑制增殖。从处理和未处理的DU-145细胞中分离RNA进行定量RT-PCR，观察PTCH、SMO、GLI1和GLI2的表达减少。为了进一步开发HH/PTCH抑制剂，我们设计了针对SMO蛋白的TM结构域和细胞内环的肽。SMO是g蛋白偶联受体（GPCR）超家族的成员，其他GPCR已被这种方法靶向。虽然针对几个TM结构域的肽对细胞增殖没有影响，但针对胞内环2和3的肽是有效的抑制剂。与环巴胺一样，活性肽导致HH/PTCH靶基因表达减少。环巴胺和SMO肽都通过诱导细胞凋亡起作用，通过TUNEL测定。为了将这些试剂推向临床研究，我们进行了生物分布分析。将放射性标记肽注射到小鼠体内，发现该肽分布在全身。目前正在计划进行疗效研究。我们还描述了SMO基因启动子区域的甲基化。在SMO高表达的细胞系中，启动子区域未甲基化；而启动子在低表达细胞中是甲基化的。MSMB蛋白的分析我们在DCEG的合作者发现MSMB基因的变异与前列腺癌相关。该基因编码微精原蛋白；一种在精液中发现的蛋白质，也是一种已知的前列腺癌生物标志物。其中一个相关的变异在基因的启动子区域发现，并影响预测的CREB转录因子结合位点。凝胶位移实验证实了这一预测。以人类FY、LCT和MHC位点为模型重建种群历史。密集的单核苷酸多态性数据的可用性允许理解群体遗传变化。通过研究种群中的基因变异，我们能够深入了解进化的力量。在过去5万年的人类进化过程中，由于暴露于间日疟原虫（Plasmodium vivax）， FY基因频率发生了变化。乳糖酶-苯丙素水解酶（LCT）基因单倍型的乳糖不耐症提供了不同的见解：成人乳糖耐受性的持久性是大约5000年前在中东出现的欧洲人获得的。有人提出，提供乳糖耐受性的基因组改变提供了在脱水中存活的优势。大多数欧洲血统的人表现出这种影响，而大多数亚洲人和撒哈拉以南非洲人以及美洲和太平洋岛民则没有。最后，我们发现最近主要组织相容性位点的基因频率发生了重大变化。我们通过研究这个基因座的人类亚群差异收集了最近进化变化的证据，现在开始定义MHC中可能赋予个体对病原体反应能力差异的特定基因区域。总之，我们相信，人口历史重建为我们的基因组的形成及其对遗传性人类疾病的责任提供了一个强有力的视角。

An individual's genetic background plays an important role in their susceptibility to cancer, disease progression, and response to therapy. Genetic variations can be used as markers to understand complex diseases including cancer, the immune/inflammatory response, and AIDS. The advent of the sequence of the human and other genomes and the ability to type large numbers of genetic markers has led to the need for greatly enhanced computing and analytic methodology. A genome wide association study for breast cancer In collaboration with scientists at the Memorial Sloan Kettering Cancer Institute we completed a whole genome association study of breast cancer in Ashkenazi Jewish subjects with a family history of disease. The scan identified several loci with potentially significant associations. From this data we selected loci for follow up studies in a replication cohort. We were able to confirm that the FGFR2 gene involved in breast cancer in several European American cohorts, is also associated with cancer in Ashkenazi Jewish populations. In addition a locus containing two genes, RNF146 and ECHDC1, was identified positively associated with breast cancer in Ashkenazi subjects. RNF146 was motifs in common with proteins that regulate estrogen receptor. In an assay for estrogen receptor function, RNF146 inhibited estrogen response. To further characterize this protein we expressed the product in bacterial cells. We are collaborating with David Waugh to crystallize the protein to determine its 3-dimensional structure. In addition we are collaborating with Allan Weissman to assess the predicted ubiquitin E3-like ligase activity that the protein is predicted to possess. Complement genes and Age-Related Macular Degeneration By studying variants in the complement B and C2 ( CFH , C2 ) genes we have identified association in this gene in patients with age-related macular degeneration (AMD). AMD is the leading cause of blindness in the elderly and is estimated to effect as many as of 10 million Americans. By examining the genetic variants in the BF and C2 genes significantly protective haplotypes were identified. The complement pathway is important in the response to pathogens causative for cancer. Further study of this important component of the innate immune response could lead to insight into human disease. A deletion that removes the CFHR1 gene adjacent to CFH is highly protective for AMD. We have obtained over 3000 DNAs from subjects in the Age-Related Eye Disease Study, and are typing them for the associated markers to understand the details of the association and identify additional loci. Recently published data implicated the Toll-like receptor 3 (TLR3) gene in AMD. However we have typed this variant in the AREDS cohort, and along with data from 7 other centers failed to replicate this association. Inhibitors of the Hedgehog/Patched Pathway in Cancer Stem Cells The HH/PTCH pathway has been demonstrated to be mutated in virtually all basal cell carcinomas and a portion of medulloblastomas. In addition, many tumors display ligand-dependent activation of the HH/PTCH pathway including pancreas, prostate, gastrointestinal tract, and small cell lung tumors. The steroid-like, natural product molecule cyclopamine specifically binds to SMO, the downstream regulator of the HH/PTCH pathway. Cyclopamine has been shown to inhibit the growth of HH/PTCH-activated cell lines and xenografts. To demonstrate whether cyclopamine inhibits the expression of HH/PTCH target genes we treated prostate (DU-145, LnCaP), gastric (AGS), and breast cancer (SK-BR3) cell lines and confirmed that cyclopamine inhibits proliferation. RNA isolated from treated and untreated DU-145 cells was subjected to quantitative RT-PCR and a reduction in expression of PTCH, SMO, and GLI1, and GLI2 was observed. To further the development of HH/PTCH inhibitors we designed peptides against TM domains and intracellular loops of the SMO protein. SMO is a member of the G-protein-coupled receptor (GPCR) superfamily, and other GPCRs have been targeted by this approach. While peptides against several TM domains failed to have an effect on cell proliferation, peptides against intracellular loops 2 and 3 were potent inhibitors. As with cyclopamine, the active peptides result in a decrease in expression of HH/PTCH target genes. Both cyclopamine and SMO peptides act by eliciting apoptosis, as measured by the TUNEL assay. To move these reagents forward into clinical studies we have performed biodistribution analysis. Radiolabelled peptide was injected into mice and the peptide was found to distribute throughout the body. Efficacy studies are now planned. We have also characterized the methylation of the promoter region of the SMO gene. In cell lines with high expression of SMO, the promoter region is unmethylated; whereas the promoter is methylated in low expressing cells. Analysis of the MSMB protein Our collaborators in DCEG identified variants in the MSMB gene as associated with prostate cancer. This gene encodes microseminoprotein, beta; a protein found in seminal fluid and a known prostate cancer biomarker. One of the associated variants is found in the promoter region of the gene, and affects a predicted CREB transcription factor binding site. Gel shift experiments confirm this prediction. Population history reconstructions using the human FY, LCT and MHC loci as models. The availability of dense single nucleotide polymorphism data permits an understanding of population genetic changes. By studying gene variants in populations we are able to provide insight into the evolutionary forces. Gene frequencies at FY have altered over the past 50,000 years of human evolution as a consequence of exposure to the malarial parasite, Plasmodium vivax. Lactose intolerance attributed to haplotypes at the Lactase-Phlorizin Hydrolase (LCT) gene provides a different insight: Adult persistence of lactose tolerance is a European acquisition arising in the middle-east about 5000 years ago. It is proposed that the genome alterations that provide for lactose tolerance provided an advantage in surviving dehydration. Most people of European ancestry show the effects of this while most Asians and sub-Saharan Africans and native peoples of the Americas and Pacific Islanders Pacific do not. Finally, we have found that there have been major recent shifts in the gene frequencies in the major histocompatibility locus. We have gathered evidence of recent evolutionary change through the study of human population sub-group differences at this locus, and are now beginning to define the specific gene regions within the MHC that may confer differences in individual ability to respond to pathogens. In sum, we believe that population history reconstruction provides a powerful lens on the shaping of our genome, and its liability for heritable human disease.