Gene Discovery in Familial Keratoconus

描述(由申请人提供)： 我们的长期目标是识别和表征圆锥角膜发病机制中涉及的基因。圆锥角膜是最常见的角膜扩张性疾病，起病于青春期。它的特点是角膜逐渐变薄，变薄的角膜突出，导致高度近视和不规则散光。它被列为一个重要的公共卫生问题 并被国家卫生研究所-国家眼科研究所列为研究重点。包括连锁分析在内的家系和双胞胎研究表明，遗传因素对圆锥角膜的发病有一定作用。VSX1基因和miR-184的突变已经被发现，但只占一小部分患者。圆锥角膜的大部分基因突变仍有待鉴定。我们假设，编码区的突变是大多数家族性患者的原因。在我们的初步研究中，我们已经收集了22个来自沙特阿拉伯的具有高度血缘关系的多人家庭。所有患者都接受了这两个已知基因的突变筛查。我们对来自两个家系的四名患者进行了完整的外显子组测序，对两个家系中不到50个候选基因进行了筛选和优先排序。在目标1中，我们将继续在其他家族中进行外显子全序列测定。我们将使用Illumina BeadChips进行纯合作图，这是在近亲交配群体中发现突变的一种强大方法。除了来自沙特阿拉伯的家庭，我们还将研究杜克大学眼科中心新入学的多元化家庭。我们预计每年通过杜克眼科中心从当地已确诊的1200多名患者中收集5-6个多重家庭和40-50名零星患者。我们每月为患者举办免费活动“圆锥角膜连接：教育同行小组会议”，这将进一步确保我们临床确诊的成功。在目标2中，我们将确定这些家族中由于DNA拷贝数变化(即基因组缺失或复制)而导致的基因突变。我们的理由基于两条证据。第一个连锁基因-chr5q31的缺失和重复与圆锥角膜有关。其次，唐氏综合征患者圆锥角膜的患病率显著增加(21号染色体的三个副本)，这也表明DNA复制的作用。我们将分析来自Aim1的Illumina BeadChips的数据，以检查DNA的缺失和复制。家庭隔离以及人类角膜中的基因表达将被用来确定候选DNA缺失和复制的优先顺序。我们将使用实时聚合酶链式反应来验证选定的候选人。在Aim1和AIM2中发现的突变将在一个包含140个多基因家系、980个零星病例和3700个受检对照的组合数据集中得到验证和复制。总之，成功完成后，我们将识别家族性圆锥角膜的基因突变，从而显著提高我们对这种疾病的了解，并为诊断和治疗提供新的靶点。

DESCRIPTION (provided by applicant): Our long term goal is to identify and characterize the genes involved in the pathogenesis of keratoconus. Keratoconus is the most common corneal ectatic disorder with onset at puberty. It is characterized by progressive corneal thinning and protrusion of the thinned corneal, which leads to high myopia and irregular astigmatism. It is listed as a significant public health concern and a research priority by the National Institute of Health - National Eye Institute. Family and twin studies including linkage analyses indicate the genetic contribution to the pathogenesis of keratoconus. Mutations in the VSX1 gene and miR-184 have been identified, but only account for a small number of patients. The majority of genetic mutations in keratoconus remain to be identified. We hypothesize that mutations in coding regions account for most of the familial patients. In our preliminary studies, we already collected 22 multiplex families from Saudi Arabia with a high degree of consanguinity. All the patients were screened for mutations in the two known genes. We have performed whole exome sequencing in four patients from two families, filtered and prioritized variants to less than 50 candidates in both families. In Aim 1, we will continue to perform whole exome sequencing in other families. We will perform homozygosity mapping using Illumina BeadChips, a powerful approach to mutation discovery in inbred populations. In addition to the families from Saudi Arabia, we will study multiplex families newly enrolled at Duke University Eye Center. We expect to collect 5-6 multiplex families and 40-50 sporadic patients per year through Duke Eye Center, from over 1,200 already diagnosed patients locally. We have a free monthly event "Keratoconus Connection: Education Peer Group Session" with patients, which will further ensure the success of our clinical ascertainment. In Aim 2, we will identify genetic mutations due to DNA copy number changes (i.e. genomic deletion or duplications) in these families. Our rationale is based on two lines of evidence. First deletions and duplications in a linkage locus- chr5q31 have been associated with keratoconus. Second, significantly increased prevalence of keratoconus in Down syndrome patients (three copies of chromosome 21) also indicates the role of DNA duplication. We will analyze data from Illumina BeadChips in Aim1 to examine DNA deletions and duplications. Family segregation as well as gene expression in human cornea will be used to prioritize the candidate DNA deletions and duplications. We will use Realtime PCR to validate the selected candidates. Mutations identified in Aim1 and Aim2 will be validated and replicated in a combined dataset with 140 multiplex families, 980 sporadic cases and 3700 examined controls. In summary, upon successful completion, we will identify genetic mutations in familial keratoconus, thus significantly improving our understanding of the disease and providing novel targets for diagnosis and treatment.