Genetic Analysis of Hyperoxia Induced Acute Lung Injury

描述（由申请人提供）：急性肺损伤（ALI）及其最严重的表现急性呼吸窘迫综合征（ARDS），代表了一种复杂和破坏性疾病的全谱，相关死亡率徘徊在30-40%之间。即使是补充氧气，这类患者的常规和必要的治疗，也会引起肺损伤。事实上，O2的有害作用已经将高氧性急性肺损伤（HALI）作为实验动物研究呼吸窘迫综合征的原型。为了应对ALI的高死亡率，并评估与现有策略不同的HALI遗传复杂性，我们建立了小鼠模型（敏感的C57BL/6J和耐药的129X1/SvJ小鼠），其长期目标是确定影响品系生存差异的基因和相关病理机制。对4种可能杂交产生的840只F2小鼠的分离分析证实，存活时间是一个复杂的性状，具有显着的外显率，以及显著的性别、杂交和亲本来源效应。对840只F2小鼠的数量性状位点（QTL）分析发现，F2种群中有三个高度显著的位点（命名为Shali1-3，用于高氧急性肺损伤的存活）和一个显著的位点（Shali4），以及一个显著的雄性特异性位点（Shali5）。两两分析确定了qtl之间的几个基因-基因相互作用以及与其他非连锁位点的上位性相互作用。分离和QTL分析表明，抗性等位基因来自亲本菌株，并重新组合以确定个体对HALI的易感性。这些结果导致了以下假设：Shali qtl包含易感基因，单独和/或在适当的等位基因组合中组合在一起时，将显著影响HALI存活时间。该应用程序的主要目的是为物理定位和数量性状基因鉴定奠定基础，主要关注沙里1。为了实现这一目标，我们提出了3个具体目标：1)在B-S模型中，通过构建与HALI存活时间显著相关的5个Shali QTL的互基因菌株，在体内确认QTL结果；确定哪些QTL对HALI存活时间有显著贡献；2)对候选基因进行检测，将Shali1 QTL区间降低到适合物理定位的水平；优先考虑和批判性地评估候选基因的功能意义；同时，通过构建和检测同源亚株，缩短Shali1 QTL间隔；3)确定在含有相应菌株对应qtl的多同源菌株中提高和降低存活率的最佳等位基因组合；在同一菌株中产生所有四个敏感或所有四个抗性沙利等位基因的互惠基因。从这些研究中获得的小鼠系将为我们提供必要的工具来识别和表征影响HALI存活的关键基因。公共卫生相关性：在我们建立的小鼠模型中使用>95%的氧气（高氧），该项目的长期目标是确定参与急性肺损伤存活的关键基因，这将使我们未来的工作重点放在所涉及的分子机制上。我们之前的遗传分析确定了五个与生存时间显著相关的染色体区域；这些区域被命名为Shali1-5，即生存至高氧急性肺损伤位点1-5。本应用程序的主要目的是通过在固定背景下生成和测试含有易感等位基因的遗传改良小鼠模型，为识别控制生存的主要基因奠定基础。

DESCRIPTION (provided by applicant): Acute lung injury (ALI) and its most severe presentation acute respiratory distress syndrome (ARDS), represent a full spectrum of a complex and devastating illness, with associated mortality hovering at 30-40%. Even supplemental O2, a routine and needed therapy for such patients, paradoxically causes lung injury. In fact, detrimental effects of O2 have established hyperoxic acute lung injury (HALI) as a prototype to study respiratory distress syndromes in experimental animals. To confront the high ALI mortality rate and to assess the genetic complexity of HALI differently than current strategies, we have established a mouse model (sensitive C57BL/6J and resistant 129X1/SvJ mice), with a long-term goal is to identify genes and the related pathologic mechanisms affecting strain survival differences. Segregation analysis of 840 F2 mice generated from the four possible intercrosses between these strains verified that survival time is a complex trait with reduced penetrance, and significant sex, cross, and parent-of-origin effects. Quantitative trait locus (QTL) analyses of the 840 F2 mice identified three highly significant loci (named Shali1-3, for Survival to hyperoxic acute lung injury) and one significant locus (Shali4) in the total F2 population, and a significant male-specific locus (Shali5). Pairwise analysis identified several gene-gene interactions among the QTLs and an epistatic interaction with an otherwise unlinked locus. Segregation and QTL analyses revealed that resistance alleles originate from both parental strains and recombine to determine individual HALI susceptibility. These results have led to the following hypothesis: Shali QTLs contain susceptibility genes that, separately and/or when grouped together in appropriate allelic combinations, will significantly affect HALI survival time. The primary objective of this application is to set the stage for physical mapping and quantitative trait gene identification, with a major focus on Shali1. To accomplish this, we propose 3 Specific Aims: 1) confirm QTL results in vivo by constructing reciprocal congenic strains for the five Shali QTLs significantly linked to HALI survival time in the B-S model; establish which QTL(s) significantly contribute to HALI survival time; 2) test candidate genes and reduce the Shali1 QTL interval to a level amenable to physical mapping; prioritize and critically assess candidate genes for functional significance; concurrently, reduce the Shali1 QTL interval by constructing and testing congenic substrains; and 3) determine the best allelic combinations for increased and decreased survival in multi-congenic strains containing the corresponding QTLs in the appropriate strain; generate reciprocal congenics with all four sensitive or all four resistant Shali alleles in the same strain. Mouse lines derived from these studies will give us the needed tools to identify and characterize the key gene(s) affecting HALI survival. PUBLIC HEALTH REVELANCE: Using >95% oxygen (hyperoxia) in our established mouse model, the long-term goal of this project is to identify critical genes involved in acute lung injury survival, which will allow us to focus future efforts on the molecular mechanisms involved. Our previous genetic analyses identified five chromosomal regions significantly linked with survival time; these regions were named Shali1-5, for Survival to hyperoxic acute lung injury loci 1-5. The primary objective of this application is to set the stage for identifying the major gene(s) controlling survival by generating and testing genetically-refined mouse models containing susceptibility alleles in a fixed background.