De novo CNV formation in vivo with sickle cell anemia therapy

描述(由申请人提供)： 镰状细胞病(SCD)是第一种在人类中发现的分子遗传疾病，影响着超过50,000美国人和全球数百万人。羟基脲(HU)是目前唯一被批准用于成人SCD的疾病修正疗法，目前正处于治疗受影响婴儿和儿童的后期临床试验阶段。HU刺激胎儿血红蛋白的产生，因此在改善氧气转运的同时降低血管闭塞危象的发生率和严重程度是有效的。然而，HU也是一种已知的复制抑制物、诱变剂和破碎剂，但HU在处理对象的后代和孙辈中的遗传效应尚未被研究，特别是在基因组水平上复制应激的影响，如拷贝数变异(CNV)突变。CNV是正常遗传变异和进化的关键因素，也是遗传疾病中常见和重要的一类突变，包括智力低下、自闭症、精神分裂症和许多其他疾病。我们已经开发了一个人类细胞培养模型系统来研究CNV突变的遗传和环境风险因素。我们发现，在与治疗患者的血清浓度相同的浓度下，HU显著诱导正常人类细胞中的CNV。这些发现对接受HU治疗的SCD和其他疾病患者的子女和孙辈的有害的、从头开始的CNV风险具有重要和直接的遗传学意义。我们建议将这些发现扩展到直接在活体动物模型中进行研究。我们将通过使用高分辨率基因组微阵列检测处理小鼠的F1和F2代来评估HU在雄性和雌性生殖系中诱导CNV的遗传效应。我们将确定新生CNV的亲代来源，检查它们的基因组结构以推断细胞和机制来源，并将研究结果与研究组动物的表型结果进行比较。此外，我们还将评估产前用HU和对照组处理的小鼠细胞和组织中CNV的体细胞嵌合性。这些研究将首次使用复制抑制模型在哺乳动物体内测试CNV的体细胞突变/复制应激假说。所获得的结果将对确定人类生殖系和躯体有害CNV的遗传和环境风险因素具有重要意义。此外，它们将对大量SCD患者和未来几代接受HU治疗的患者具有重要和直接的临床意义。

DESCRIPTION (provided by applicant): Sickle cell disease (SCD) is the first molecular genetic disorder identified in humans, affecting over 50,000 Americans and millions of people worldwide. Hydroxyurea (HU) is currently the only approved disease- modifying therapy for adult SCD and is in late-stage clinical trials for treatment of affected infants and children. HU stimulates fetal hemoglobin production and is therefore effective in improving oxygen transport while reducing the incidence and severity of vaso-occlusive crises. However, HU is also a known replication inhibitor, mutagen and clastogen, yet the genetic effects of HU in the offspring and grandchildren of treated subjects have not been studied, particularly at the genomic level for effects of replication stress such as copy number variant (CNV) mutations. CNVs are a key factor in normal genetic variation and evolution and are a common and important class of mutation in genetic disorders, including mental retardation, autism, schizophrenia and many others. We have developed a human cell culture model system to investigate the genetic and environmental risk factors for CNV mutations. We have found that HU, at concentrations identical to serum concentrations in treated patients, significantly induces CNVs in normal human cells. These findings have important and direct genetic implications for deleterious, de novo CNV risk in the children and grandchildren of patients treated with HU for SCD and other disorders. We propose to extend these findings to direct studies in animal models in vivo. We will evaluate the genetic effects of HU on CNV induction in both the male and female germlines by examining F1 and F2 generations of treated mice using high-resolution genomic microarrays. We will determine parental origins of de novo CNVs, examine their genomic structures to infer cellular and mechanistic origins and compare findings with those from phenotyping of study group animals. In addition, we will assess somatic mosaicism for CNVs in cells and tissues of mice treated prenatally with HU and controls. These studies will provide the first in vivo test in mammals of the somatic mutation/replication stress hypothesis for CNVs using a model inhibitor of replication. The results obtained will have important implications for defining genetic and environmental risk factors for both germline and somatic deleterious CNVs in humans. Moreover they will have important and immediate clinical significance to a large number of individuals with SCD and the future generations of HU-treated patients.