New Gene Discoveries and Biology of Ribosomes in Diamond-Blackfan Anemia

描述（由申请人提供）：Diamond-Blackfan贫血（DBA）是已知由核糖体蛋白（RP）基因突变引起的第一种人类疾病。其特征是贫血、身体异常和恶性肿瘤风险增加。迄今为止，在约53%的DBA患者中报告了9种RP基因（RPS 19、RPS 24、RPS 17、RPL 35 A、RPL 5、RPL 11、RPS 7、RPS 10和RPS 26）的突变。最近，我们完成了所有80个核糖体蛋白基因的筛选，并获得了两个额外的RP基因RPL 19和RPL 26突变的证据，约2%的DBA患者。尽管在破译DBA的遗传原因方面取得了重大进展，但这些患者的诊断和遗传咨询仍然受到大约50%的病例具有尚未确定的基因突变的严重阻碍。这种确认的遗传病因学的缺乏也使得难以建立合适的动物模型， DBA，并对疾病的机制进行进一步的研究。为了解决这个问题，我们正在对来自DBA先证者的70个DNA样本进行比较基因组杂交，这些DNA样本没有已知的突变，以寻找我们已经测序的80个RP基因和参与核糖体生物发生的~120个基因中的缺失和重复。我们还建议进行全外显子组测序（“下一代”测序）的30个DNA样本的DBA先证者谁是已知的DBA基因筛选和这些基因突变阴性。比较基因组杂交将使我们能够识别RP和其他候选基因中的微缺失或重复;全外显子组测序（所有外显子和内含子-外显子边界的测序）将使我们能够识别其他DBA基因，我们假设这些基因可能编码参与核糖体生物发生或功能的蛋白质。为了进一步验证我们的假设，即异常核糖体生物发生是所有患者DBA机制的基础，我们还将对来自新发现基因突变患者的淋巴母细胞系的RNA样本进行前rRNA成熟测定。我们的假设预测，异常成熟的前rRNA将是一个共同的特点，在患者与新发现的基因。我们还假设DBA的临床异质性是遗传变异的结果，遗传变异影响相关先天性出生缺陷的存在或患者对类固醇治疗的反应。研究这些变异的影响将扩大我们对DBA机制的理解，特别是与类固醇反应的相关性，可能成为DBA靶向治疗的平台。以下三个具体的目标将使我们能够找到DBA的遗传基础和贫血的分子机制在这种疾病。具体目标1。在已知DBA基因无突变的患者中鉴定新的DBA基因。具体目标2。确定DBA基因在前RNA成熟中的作用。具体目标3。通过全基因组关联研究确定DBA中的修饰基因。 公共卫生相关性：Diamond-Blackfan贫血是已知的第一种由核糖体蛋白基因突变引起的人类疾病。其特征是贫血、身体异常和恶性肿瘤风险增加。治疗的选择是类固醇治疗，但这有显着的副作用。为了更好地治疗这些患者，了解遗传基础和了解疾病的机制是非常重要的。该项目的目标是在大约50%没有已知突变的患者中鉴定基因，并探索核糖体蛋白在Diamond-Blackfan贫血发病机制中的作用，这将为更好的治疗发现奠定基础。

DESCRIPTION (provided by applicant): Diamond-Blackfan anemia (DBA) is the first human disease known to be caused by mutations in ribosomal protein (RP) genes. It is characterized by anemia, physical anomalies, and increased risk of malignancy. To date mutations in nine RP genes, RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10 and RPS26 have been reported in ~53% of DBA patients. Recently, we finished screening all 80 ribosomal protein genes and have obtained evidence for mutations in two additional RP genes, RPL19 and RPL26, in ~2% of DBA patients. Despite this significant progress in deciphering the genetic causes of DBA, diagnostics and genetic counseling for these patients are still severely hampered by the fact that roughly 50% of cases have mutations in genes yet to be identified. This lack of confirmed genetic etiology also makes it difficult to create suitable animal model for DBA and to perform further studies on the mechanisms of the disease. To address this issue, we are performing comparative genomic hybridization on 70 DNA samples from DBA probands without known mutations to search for deletions and duplications in the 80 RP genes we have already sequenced and in ~120 genes involved in ribosomal biogenesis. We also propose to perform whole-exome sequencing ("next generation" sequencing) on 30 DNA samples from DBA probands who were screened for known DBA genes and are negative for mutations in these genes. Comparative genomic hybridization will allow us to identify microdeletions or duplications in RP and other candidate genes; the whole-exome sequencing (sequencing of all exons and intron-exon boundaries) will allow us to identify the additional DBA genes, which we hypothesize will likely encode proteins involved in ribosomal biogenesis or function. To further test our hypothesis that abnormal ribosomal biogenesis underlies the mechanism of DBA in all patients, we will also perform pre-rRNA maturation assays on RNA samples from lymphoblastoid cell lines from patients with mutations in the newly discovered genes. Our hypothesis predicts that abnormal maturation of pre-rRNA will be a common feature in patients with newly discovered genes. We also hypothesize that profound clinical heterogeneity in DBA is a consequence of genetic variants that influence the presence of associated congenital birth defects or patients' response to steroid treatment. Investigating the influence of these variants would expand our understanding of the mechanism of DBA and potentially, especially for the association with response to steroids, could be a platform for the targeted therapies for DBA. The following three specific aims will allow us to find the genetic basis of DBA and the molecular mechanism of anemia in this disease. Specific Aim 1. Identify novel DBA gene(s) in patients without mutations in known DBA genes. Specific Aim 2. Determine the role of DBA genes in pre-RNA maturation. Specific Aim 3. Identify modifier genes in DBA by performing a genome-wide association study. PUBLIC HEALTH RELEVANCE: Diamond-Blackfan anemia is the first human disease known to be caused by mutations in ribosomal protein genes. It is characterized by anemia, physical anomalies, and increased risk of malignancy. The treatment of choice is a steroid therapy, but this has significant side effects. In order to find better treatments for these patients it is impotant to know the genetic basis and understand the mechanism of the disease. The goal of this project, is to identify genes in approximately 50% of patients without known mutations and to explore the role of ribosomal proteins in the pathogenesis of Diamond-Blackfan anemia, which will establish the basis for better treatment discovery.