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），代表了一种复杂和毁灭性疾病的全谱，相关死亡率徘徊在30- 40%。即使是补充O2，这类患者的常规和必要的治疗，矛盾的是会导致肺损伤。事实上，O2的有害作用已经建立了高氧急性肺损伤（HALI）作为研究实验动物呼吸窘迫综合征的原型。为了面对高的ALI死亡率，并评估HALI的遗传复杂性，与目前的策略不同，我们建立了一个小鼠模型（敏感的C57 BL/6 J和耐药的129 X1/SvJ小鼠），长期目标是确定基因和影响菌株生存差异的相关病理机制。分离分析的840个F2小鼠产生的四个可能的intercrosses这些菌株之间证实，生存时间是一个复杂的性状，减少的遗传，和显着的性别，交叉，和父母的起源的影响。对840只F2小鼠进行的数量性状基因座（QTL）分析在总F2群体中鉴定出三个高度显著的基因座（命名为Shali 1 -3，用于高氧急性肺损伤的存活）和一个显著的基因座（Shali 4），以及一个显著的雄性特异性基因座（Shali 5）。成对分析确定了几个QTL之间的基因-基因相互作用和上位性相互作用，否则不连锁的位点。分离和QTL分析表明，抗性等位基因来源于两个亲本菌株和重组，以确定个人HALI易感性。这些结果导致了以下假设：Shali QTL含有易感基因，单独和/或当在适当的等位基因组合组合在一起时，将显着影响HALI存活时间。本申请的主要目的是为物理作图和数量性状基因鉴定奠定基础，主要关注Shali 1。为此，我们提出了3个具体的目标：1）通过构建B-S模型中与HALI存活时间显著连锁的5个Shali QTL的相互同源株系，在体内确认QTL结果，确定哪些QTL显著影响HALI存活时间; 2）检测候选基因，并将Shali 1 QTL区间缩小到适合物理作图的水平;对候选基因的功能意义进行优先排序和严格评估;同时，通过构建和测试同源亚株来减少Shali 1 QTL间隔;以及3）确定在适当菌株中含有相应QTL的多同源菌株中增加和减少存活的最佳等位基因组合;在同一品系中产生具有所有四个敏感或所有四个抗性Shali等位基因的相互同源基因。从这些研究中获得的小鼠品系将为我们提供鉴定和表征影响HALI存活的关键基因所需的工具。公共卫生部门：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Shali 1 -5，即高氧急性肺损伤位点1-5的生存。本申请的主要目的是通过在固定背景下生成和测试含有易感性等位基因的遗传改良小鼠模型，为鉴定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.