Clonal Hematopoiesis in Diamond Blackfan Anemia

描述(申请人提供)：钻石黑扇贫血(DBA)是一种遗传性骨髓衰竭(BMF)综合征，在婴儿期表现为红细胞再生障碍性疾病，有时伴有发育异常。与其他(BMF)综合征一样，BMF是可变的，家庭成员可以携带突变，但表现为轻微或没有BMF，或者在其他情况下，患者可以进入长期缓解。我们假设可变外显率和缓解是由于干细胞或多能祖细胞的体细胞突变，这些突变将细胞从遗传突变的影响中拯救出来，导致克隆性造血和临床缓解。我们相信，如果我们能够确定这些基因变化的性质，它们将增加我们对DBA发病机制的理解，并提出可能用于开发新治疗方法的潜在药物靶点。我们将从20名DBA患者的骨髓和皮肤活检中提取DNA，其中包括一些临床缓解期患者。我们将确定同一人的骨髓样本和皮肤样本之间的遗传差异，以确定骨髓中发生的体细胞突变。这将使用两种方法来完成：1.高密度SNP微阵列分析，以发现外显子组测序无法发现的基因组变化，包括拷贝数变化和拷贝中性杂合性丢失的获得，其中常染色体基因的两个拷贝可能来自同一亲本；2.完整外显子组测序，以识别蛋白质编码区的非同义突变。在功能研究之前，通过对第二组患者进行重新测序和生物信息学分析来评估它们对造血的可能影响，将进一步验证可能的候选挽救突变。

DESCRIPTION (provided by applicant): Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure (BMF) syndrome that presents in infancy with red cell aplasia, sometimes with developmental abnormalities. In common with other (BMF) syndromes the BMF is variable and family members can carry the mutation but show mild or no BMF or, in other cases, patients can go into long lasting remission. We hypothesize that the variable penetrance and remission are due to somatic mutations in stem cells or multipotent progenitors that rescue cells from the effects of the inherited mutation, leading to clonal hematopoiesis and clinical remission. We believe that if we can determine the nature of these genetic changes they will increase our understanding of the pathogenesis of DBA and suggest potential drug targets that may be used in developing new treatments. We will isolate DNA from bone marrow and skin biopsies from 20 DBA patients, including some in clinical remission. We will determine genetic differences between the bone marrow sample and the skin sample from the same person to identify somatic mutations that have occurred in the bone marrow. This will be done using two approaches 1. High density SNP microarray analysis, to find genomic changes that will not be found by exome sequencing including copy number changes and acquisition of copy neutral loss of heterozygosity, where both copies of autosomal genes may derive from the same parent and 2. Whole exome sequencing to identify non-synonymous mutations in protein coding regions. Prior to functional studies, likely candidates for rescuing mutations will be further validated by re-sequencing in a second group of patients and bioinformatic analysis to assess their possible effects on hematopoiesis.