Characterization of RGC death susceptibility alleles

描述（申请人提供）：青光眼是一种慢性致盲性神经退行性疾病，以视网膜神经节细胞的进行性丧失和视神经变性为特征。尽管在该病的遗传学领域已经取得了重大进展，但到目前为止，这些研究大多只研究了表现出孟德尔遗传模式的罕见形式的该病。然而，大多数青光眼是复杂的遗传疾病，有多个相互作用的基因座影响个体的易感性。一个可能的易感性领域是由垂死的神经节细胞执行的基因控制的细胞死亡程序。我们已经在小鼠身上使用实验遗传学来帮助识别可能影响这一过程的潜在易感等位基因。对15个近交系小鼠进行视神经挤压后神经节细胞损失的筛选显示，有2个系对这一过程有不同的抵抗力。发现抗性表型作为显性性状遗传，全基因组定位鉴定出负责基因位于第5染色体25 cM间隔内（Chr5.loc34-59）。这个位点被命名为视网膜神经节细胞易感基因1 （Rgcs1）。我们建议通过继续精细绘制Rgcs1位点来缩小感兴趣的区域来扩展这些观察结果。精细制图将开始使用SNP分析将区间细化到预测的10 cM区域，然后生成区间特定基因系（ISCLs）将该区域分解为1 cM重叠区间。此外，当区间变小时，我们将使用计算机映射来帮助定义感兴趣的候选基因。迄今为止，我们已经使用这种方法从已知存在于该区域的578个基因中确定了7个潜在的候选基因。候选基因将在两个亲本近交系之间进行定量和定性分析。在产生iscl的过程中，我们还将创建DBA/2J小鼠的亚株，以研究Rgcs1位点在青光眼易感性中的作用。野生型DBA/2J小鼠携带该抗性等位基因。有趣的是，这些小鼠也会患上慢性遗传性青光眼，这似乎是一个悖论，但也可能与真正的易感性等位基因相一致。我们测试Rgcs1在青光眼中的作用，将从BALB/cByJ动物中易感的Rgcs1位点交叉到DBA/2J背景中，产生子菌株DBA/2J. balbrgcs1。我们预计这些小鼠会出现眼压升高，并且由于携带易感的Rgcs1等位基因，会出现更严重的青光眼。最后，随着我们获得更多关于小鼠Rgcs1位点的信息，我们将开始通过使用杜克大学人类遗传学中心的人类青光眼患者数据集检查关键候选基因的SNP差异来询问人类基因组的合成区。本提案描述了旨在识别和表征位于小鼠5号染色体区域的基因的研究，该基因影响视神经挤压后视网膜神经节细胞的存活。我们还将在小鼠青光眼模型中测试该染色体区域影响神经节细胞存活的能力，然后扩展这些观察，以确定相应的位点/基因是否影响人类青光眼。这项工作的意义在于它将有助于阐明与青光眼复杂性状相关的一些易感等位基因。发现和描述这些等位基因将有助于青光眼患者更好的诊断和治疗方案。

DESCRIPTION (provided by applicant): Glaucoma is a chronic blinding neurodegenerative disease characterized by the progressive loss of retinal ganglion cells and degeneration of the optic nerve. Although significant progress has been made in the field of the genetics of this disease, the majority of these studies have so far examined rarer forms of the disease that exhibit Mendelian inheritance patterns. The majority of glaucomas, however, are complex genetic diseases that have multiple interacting loci that affect an individual's susceptibility. One possible area of susceptibility is the genetically controlled cell death program that is executed by dying ganglion cells. We have used experimental genetics in mice to help identify potential susceptibility alleles that could affect this process. A screen of 15 inbred mouse lines for the amount of ganglion cell loss after optic nerve crush revealed 2 lines with varying resistance to this procedure. The resistant phenotype was found to be inherited as a dominant trait, and genome wide mapping has identified the responsible gene to be located within a 25 cM interval of chromosome 5 (Chr5.loc34-59). This locus has been named Retinal ganglion cell susceptible 1 (Rgcs1). We propose to extend these observations by continuing to fine map the Rgcs1 locus to narrow the region of interest. Fine mapping will begin using SNP analysis to refine the interval to a predicted 10 cM region, followed by the generation of Interval Specific Congenic Lines (ISCLs) to dissect the region into 1 cM overlapping intervals. In addition, we will use in silico mapping to help define candidate genes of interest in the interval as it becomes smaller. To date, we have used this approach to identify 7 potential candidates from among the 578 genes known to exist in this region. Candidate genes will be characterized by quantitative and qualitative analyses between the two parental inbred strains. During the course of generating ISCLs, we will also create a substrain of DBA/2J mice to examine the role that the Rgcs1 locus plays in susceptibility to glaucoma. Wild type DBA/2J mice carry the resistant allele. Interestingly, these mice also develop chronic inherited glaucoma, which may seem like a paradox, but could also be consistent with a true susceptibility allele. Our test of the role of Rgcs1 in glaucoma, will be to cross the susceptible Rgcs1 locus from BALB/cByJ animals onto the DBA/2J background to generate the substrain DBA/2J.BALBRgcs1. We expect these mice to develop elevated intraocular pressure, and by virtue of carrying a susceptible Rgcs1 allele, a more severe form of glaucoma. Lastly, as we acquire more information on the mouse Rgcs1 locus, we will begin to interrogate the syntenic region of the human genome by examining for SNP differences in key candidate genes using a data set of human glaucoma patients housed at the Center for Human Genetics at Duke University. PUBLIC HEALTH RELEVANCE This proposal describes studies designed to identify and characterize a gene located in a region of mouse chromosome 5 that affects retinal ganglion cell survival after optic nerve crush. We will also test the ability of this chromosomal region to affect ganglion cell survival in a mouse model of glaucoma, and then extend these observations to determine if the corresponding locus/gene affects glaucoma in humans. The significance of this work is that it will help elucidate some of the susceptibility alleles associated with the complex trait of glaucoma. Finding and characterizing these alleles will lead to better diagnosis and treatment regimes for individuals with glaucoma.