New gene discoveries and biology of ribosomes in Diamond-Blackfan anemia.

描述（由申请人提供）：Diamond-Blackfan贫血（DBA）是已知的第一个由核糖体蛋白（RP）基因突变引起的人类疾病。它的特点是贫血，身体异常，恶性肿瘤的风险增加。迄今为止，约53%的DBA患者报告了9种RP基因RPS19、RPS24、RPS17、RPL35A、RPL5、RPL11、RPS7、RPS10和RPS26的突变。最近，我们完成了所有80个核糖体蛋白基因的筛选，并在约2%的DBA患者中获得了另外两个RP基因RPL19和RPL26突变的证据。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严重阻碍，因为大约50%的病例存在尚未确定的基因突变。由于缺乏确定的遗传病因，也给DBA建立合适的动物模型和进一步研究其发病机制带来了困难。为了解决这个问题，我们正在对来自DBA先知者的150个DNA样本进行比较基因组杂交，以寻找我们已经测序的80个RP基因中的缺失和重复。我们还建议对DBA先证者的28个DNA样本进行全外显子组测序（“下一代”测序），这些样本筛选了所有80个RP基因，并且这些基因的突变均为阴性。比较基因组杂交将使我们能够识别RP和其他候选基因的微缺失或重复；整个外显子组测序（所有外显子和内含子-外显子边界的测序）将使我们能够识别额外的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细胞系的RNA样本进行预rrna成熟分析。我们的假设预测，pre-rRNA的异常成熟将是新发现基因患者的共同特征。我们还假设DBA的临床异质性是遗传变异的结果，遗传变异会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关联，可能为DBA的靶向治疗提供平台。以下三个具体目的将使我们找到DBA的遗传基础和本病贫血的分子机制。具体目标在大约50%没有已知突变的患者中确定导致DBA的新基因。具体目标2。确定DBA基因在rna前成熟中的作用。具体目标3。通过进行全基因组关联研究，确定DBA中的修饰基因。

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 150 DNA samples from DBA probands without known mutations to search for deletions and duplications in the 80 RP genes we have already sequenced. We also propose to perform whole exome sequencing ("next generation" sequencing) on 28 DNA samples from DBA probands who were screened for all 80 RP 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 the novel gene(s) causing DBA in about 50% of patients without known mutations. 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.