Pathogenetics of the FOX transcription factor gene cluster on 16q24.1

描述(申请人提供)：肺泡毛细血管发育不良伴肺静脉错位(ACD/MPV)是一种致命的新生儿弥漫性肺部发育障碍，通常与涉及心脏、胃肠道和泌尿生殖系统的多种先天性异常有关。受ACD/MPV影响的婴儿在出生后的头几天会出现严重的呼吸窘迫和肺动脉高压，尽管接受了重症监护，但他们通常很快就会死亡。最近，我们发现由于转录因子Forkhead Box F1(FOXF1)在16q24.1上的点突变或基因组缺失而导致的单倍性不足导致ACD/MPV和广泛的先天性畸形。此外，我们还发现了FOXF1上游的两个不同的微缺失，暗示了位置效应在疾病的发病机制中的作用。在FOXF1微缺失中遇到的多效性效应，如左心发育不良综合征和胃肠道失调，可能是由于邻近基因FOXC2和FOXL1的单倍性不足所致，这两个基因都是位于16q24.1的Fox簇的一部分。杂合子Foxf1小鼠死于肺出血，并伴有严重的肺泡化和血管生成缺陷以及其他器官异常，尽管它们不能完全概括人类的ACD/MPV。Foxf1基因在发育过程中的表达表明了一种有趣的基因调控模式。我们推测，Foxf1的这种复杂调控可能是由于位置效应和基因组印记在组织和时间特异性的方式；ACD/MPV也可以由其他基因(S)或FOXF1调节元件的破坏引起；通过增加Foxf1蛋白在毛细血管内皮细胞和周围间充质中的剂量，可以在围生期预防Foxf1小鼠的肺缺陷。我们设计了三个目标来检验这些假设。在目标1中，我们将从两个方面剖析FOXF1的基因调控。首先，将使用报告分析、芯片上芯片和染色质构象捕获(3C)技术识别和测试可能对Fox基因簇的表达或FOXF1特定表达重要的调控元件。其次，我们将分析Foxf1在小鼠体内的分离和等位基因特异性表达。在目标2中，我们将使用我们在目标1中的研究获得的知识来筛选FOXF1上游或下游的调控元件以及ACD/MPV候选基因中的点突变和拷贝数变异。最后，在目标3中，我们将通过使用腺病毒载体基于腺病毒载体的Foxf1基因在小鼠外周肺转移来探索治疗方案。ACD/MPV是一种致命的疾病，目前还没有可用的治疗方法。我们相信，使用病毒载体的基因治疗方法可能通过逆转致死性毛细血管缺陷的异常形成来纠正ACD/MPV患者的致死性表型。此外，与这种实验性疗法相关的风险可能从风险的角度来证明是合理的：从好处的角度来看，并且有可能在医院环境中被转化。

DESCRIPTION (provided by applicant): Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a lethal neonatal diffuse developmental disorder of the lungs, which is commonly associated with multiple congenital anomalies involving the cardiac, gastrointestinal, and genitourinary systems. Infants affected with ACD/MPV develop severe respiratory distress with pulmonary hypertension within the first few days of life and despite intensive care they usually die soon thereafter. Recently, we have found that haploinsufficiency due to point mutations or genomic deletions of the transcription factor Forkhead Box F1 (FOXF1) on 16q24.1 results in ACD/MPV and a broad spectrum of congenital malformations. In addition, we have identified two distinct microdeletions upstream of FOXF1, implicating a position effect in the pathogenesis of the disease. Pleiotropic effects encountered in FOXF1 microdeletions, such as hypoplastic left heart syndrome and gastrointestinal atresias, may be due to haploinsufficiency for the neighboring genes, FOXC2 and FOXL1, both part of the FOX cluster at 16q24.1. Heterozygous Foxf1 mice die from pulmonary hemorrhage with severe defects in lung alveolarization and vasculogenesis along with other organ anomalies, although they do not completely recapitulate ACD/MPV in humans. The expression of the Foxf1 gene during development suggests an intriguing pattern of gene regulation. We hypothesize that this complex regulation of Foxf1 may be due to both position effects and genomic imprinting in a tissue- and time-specific manner; ACD/MPV can also be caused by disruption of other gene(s) or FOXF1 regulatory elements; and the lung defect in Foxf1 mice can be prevented perinatally by increasing the dosage of the Foxf1 protein in the capillary endothelium and surrounding mesenchyme. We have designed three aims to test these hypotheses. In aim 1, we will dissect gene regulation of FOXF1 in two ways. First, regulatory elements that may be important to the expression of the FOX gene cluster or to FOXF1 specific expression will be identified and tested using reporter assays, ChIP-on-chip, and chromatin conformation capture (3C) techniques. Second, we will analyze the segregation and allele-specific expression of Foxf1 in mice. In aim 2, we will use the knowledge gained from our studies in aim 1 to screen for point mutations and copy-number variations in the regulatory elements identified upstream or downstream to FOXF1 and in the ACD/MPV candidate genes. Finally, in aim 3, we will explore therapeutic options by use of adenoviral vector-based Foxf1 gene transfer in peripheral murine lungs. ACD/MPV is a lethal disorder and there is no available treatment at the present time. We believe that a gene therapy approach using viral vectors may have the potential to correct the lethal phenotype of ACD/MPV patients by reversing the abnormal formation of the lethal capillary defect. Moreover, the risks related to this experimental therapy may be justified from a risk: benefit standpoint and have potential to be translated in the hospital setting.