Genetic Analysis of Hyperoxia Induced Acute Lung Injury

描述(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)，代表了一种复杂和毁灭性疾病的全过程，相关死亡率徘徊在30%-40%。即使是补充氧气，这是这类患者的常规和必要的治疗，也会矛盾地导致肺损伤。事实上，氧气的有害作用已经建立了高氧性急性肺损伤(HALI)作为研究实验动物呼吸窘迫综合征的原型。为了应对ALI的高死亡率，并以不同于现有策略的方式评估HALI的遗传复杂性，我们建立了一个小鼠模型(敏感的C57BL/6J和抗性的129X1/SVJ小鼠)，长期目标是识别影响菌株生存差异的基因和相关的病理机制。对这些品系之间四个可能的杂交产生的840只F2小鼠进行的分离分析证实，生存时间是一个复杂的性状，具有降低的外显性，以及显著的性别、杂交和亲本效应。对840只F2小鼠进行数量性状基因座(QTL)分析，在F2群体中发现了3个对高氧急性肺损伤存活有极显著影响的基因座(命名为Shali1-3)和1个显著基因座(Shali4)，以及1个显著的雄性特有基因座(Shali5)。配对分析确定了QTL之间的几个基因-基因交互作用，以及与另外一个不连锁的基因座的上位性交互作用。分离和QTL分析表明，抗性等位基因来源于两个亲本菌株，并通过重组决定了个体对赤霉病的敏感性。这些结果导致了以下假设：沙利QTL含有感病基因，如果单独和/或以适当的等位基因组合组合在一起，将显著影响哈利的存活时间。这一应用的主要目标是为物理作图和数量性状基因鉴定奠定基础，重点是Shali1。为此，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个沙力QTL的互反同源品系，在体内确认QTL结果；确定哪个QTL(S)显著影响HALI存活时间；2)测试候选基因，并将沙力1的QTL区间缩小到符合物理作图的水平；优先考虑并严格评估候选基因的功能意义；同时，通过构建和测试同源亚系，缩短沙里1的QTL间隔；以及3)确定在适当品系中含有相应QTL的多个同源品系的最佳等位基因组合；在同一品系中产生与所有四个敏感或所有四个抗性沙利等位基因互惠的同源基因。来自这些研究的小鼠品系将为我们提供必要的工具来识别和表征影响HALI生存的关键基因(S)。公共卫生研究：在我们已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识别与急性肺损伤存活相关的关键基因，这将使我们能够将未来的努力集中在相关的分子机制上。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Survival to Hyperoxic急性肺损伤基因座1-5。这项应用的主要目标是通过在固定背景下生成和测试包含易感等位基因的经过遗传改进的小鼠模型，为识别控制生存的主要基因(S)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.