Gene Discovery in Familial Keratoconus

描述（由申请人提供）： 我们的长期目标是确定和表征与圆锥角膜发病机制有关的基因。圆锥角膜是青春期最常见的角膜扩张性疾病。其特征在于进行性角膜变薄和变薄的角膜突出，从而导致高度近视和不规则散光。它被列为一个重大的公共卫生问题 也是国家健康研究所-国家眼科研究所的研究重点。家系和双生子研究包括连锁分析表明圆锥角膜的发病机制与遗传有关。VSX 1基因和miR-184的突变已被确定，但仅占少数患者。圆锥角膜中的大多数基因突变仍有待确定。我们假设编码区的突变占大多数家族性患者。在我们的初步研究中，我们已经从沙特阿拉伯收集了22个具有高度血缘关系的多重家庭。所有患者都接受了两种已知基因突变的筛查。我们对来自两个家族的四名患者进行了全外显子组测序，过滤并优先考虑两个家族中少于50个候选者的变体。在目标1中，我们将继续在其他家族中进行全外显子组测序。我们将使用Illumina BeadChips进行纯合性作图，这是一种在近交群体中发现突变的强大方法。除了来自沙特阿拉伯的家庭外，我们还将研究杜克大学眼科中心新注册的多功能家庭。我们预计每年通过杜克眼科中心从当地1,200多名已确诊的患者中收集5-6个多重家族和40-50名散发患者。我们有一个免费的每月活动“圆锥角膜连接：教育同行小组会议”与患者，这将进一步确保我们的临床确定的成功。在目标2中，我们将确定这些家族中由于DNA拷贝数变化（即基因组缺失或重复）而导致的基因突变。我们的理由基于两条证据。第一个连锁基因座chr 5 q31的缺失和重复与圆锥角膜相关。其次，唐氏综合征患者圆锥角膜患病率的显著增加（21号染色体的三个拷贝）也表明DNA复制的作用。我们将在Aim 1中分析Illumina BeadChips的数据，以检查DNA缺失和重复。家族分离以及人类角膜中的基因表达将用于优先考虑候选DNA缺失和重复。我们将使用实时PCR来验证所选择的候选人。在Aim 1和Aim 2中鉴定的突变将在具有140个多重家族、980个散发病例和3700个检查对照的组合数据集中进行验证和复制。总之，成功完成后，我们将确定家族性圆锥角膜的基因突变，从而显著提高我们对该疾病的理解，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.