High Resolution Helicobacter SNP Typing to Identify Carcinogenic cagPAI Variants

描述（由申请人提供）：拟议研究的长期目标是为制定有效的预防幽门螺杆菌（HP）相关疾病的一级预防策略提供科学依据，特别是胃癌，它仍然是全球第二大癌症死亡原因和第四大最常诊断的癌症。在美国，少数族裔，如亚洲人、黑人、西班牙裔和美洲原住民的发病率几乎是非西班牙裔白人的两倍。现在已经明确cagA和IV型分泌系统（T4SS）在hp相关疾病的发病机制中起核心作用。细胞毒素相关基因致病性岛（cagPAI）由HP基因组中一个40kb的DNA区域组成，包含大约31个开放阅读框，编码细胞毒素和T4SS的结构和功能成分，T4SS作为分子注射器将细菌大分子注射到宿主细胞细胞质中。然而，在大多数HP为cagA阳性菌株的高危人群中，仅凭cagA状态（cagPAI的标志物）不足以预测临床结果。在此，我们假设cagPAI微变异性是cagA毒性的重要决定因素，导致高级别胃癌前病变和癌性病变的风险增加或降低。该研究将采用高通量测序和基因分型技术来了解与胃组织病理学相关的HP序列变异。迄今为止，高分辨率单核苷酸多态性（SNP）分型很少用于对特定病原体进行分类，尽管它广泛应用于人类基因组。拟议的研究旨在扩大我们在HP和胃肠道病理研究方面持续成功的国际合作。我们证明了细胞毒素相关基因致病性岛（cagPAI）标记物cagA基因的存在比宿主和环境因素对高级别癌前病变的风险有更大的影响，并发现了可能改变胃癌风险的cag基因微变异。具体来说，我们建议通过在来自高风险和低风险拉丁美洲国家的2000多名cagA阳性HP感染者中检测与癌症和高级别胃癌前病变风险的统计关联，来验证我们关于cagPAI基因变异和胃癌的初步观察结果。我们还将把这种方法扩展到其他cagPAI基因，以发现新的变体。因此，本项目将重点研究cagA、cagC、cagE、cagI、cagL、cagYc和caggamma， (a)它们的功能已经在模型细菌T4SS中得到了很好的表征，和/或(b)它们存在于细胞外，表明可能与宿主细胞相互作用。与未被组织学诊断为胃高级别癌前病变或癌症的对照组相比，我们将调查所选cagPAI基因的微变异是否与胃癌风险或高级别癌前病变风险相关，并评估这些cagPAI微变异的患病率是否与参与人群中胃癌发病率的地理差异相关。

DESCRIPTION (provided by applicant): The long-term goal of the proposed study is to provide a scientific basis to develop efficient primary prevention strategies against Helicobacter pylori (HP) associated morbidities, particularly gastric cancer, which remains the 2nd leading cause of cancer death and the 4th most commonly diagnosed cancer worldwide. Within the US, ethnic minorities, e.g., Asians, Blacks, Hispanics and Native Americans, experience an incidence almost twice as high as in non-Hispanic Whites. It is now clearly established that cagA and the type IV secretion system (T4SS) play a central role in the pathogenesis of HP-associated diseases. The cytotoxin-associated gene pathogenicity island (cagPAI) consists of a 40kb DNA region in the HP genome, and contains approximately 31 open reading frames, encoding cytotoxins and structural and functional components of the T4SS that acts as a molecular syringe injecting bacterial macromolecules into host cell cytosol. However, cagA status (a marker of cagPAI) alone is not sufficient to predict clinical outcomes in high risk populations where the majority of HP is cagA positive strains. Herein we hypothesize that cagPAI microvariability is an important determinant of cagA toxicity, leading to increased or reduced risk of high grade gastric precancerous and cancerous lesions. The proposed study will employ high throughput sequencing and genotyping technologies to gain knowledge of HP sequence variants associated with gastric histopathology. To date, high resolution single nucleotide polymorphisms (SNP) typing has rarely been used to classify specific pathogens, despite its widespread application to the human genome. The proposed study is aimed to extend our sustained successful international collaboration in studies on HP and gastrointestinal pathologies. We have demonstrated that the presence of cagA gene, marker of cytotoxin- associated gene pathogenicity island (cagPAI) has much greater impact on the risk of high grade premalignant lesions in comparison with host and environmental factors and identified possible cag gene microvariants that can alter the risk of gastric cancer drastically. Specifically we propose to validate our pilot observations concerning cagPAI genetic variants and gastric cancer by testing statistical associations with risk of cancer and high grade gastric precancerous lesions in large well characterized populations totaling more than 2000 individuals infected with cagA positive HP from both high and low risk Latin American countries. We will also expand this approach to additional cagPAI genes to discover new variants. Consequently this project will focus on cagA, cagC, cagE, cagI cagL, cagYc and cag Gamma, (a) whose functions have been well characterized in the model bacterial T4SS and/or (b) which are present extracellularly, suggesting possible interactions with host cells. We will investigate whether microvariants in the selected cagPAI genes are associated with gastric cancer risk or risk of high grade premalignant lesions, compared with the control group of the subjects who were not histologically diagnosed with gastric high grade premalignant lesions or cancer, and evaluate if the prevalence of those cagPAI microvariants are correlated with geographic variation in gastric cancer incidence within participating populations.