Etiology and pathogenesis of lethal lung developmental disorders in neonates

项目摘要 致死性肺发育障碍（LLDD）很少被诊断，但毁灭性的肺发育不全 (PHs)表现为进行性新生儿缺氧和重度肺动脉高压（PAH）。基于 根据组织病理学表现，LLDD传统上被分类为肺泡毛细血管发育不良， 肺静脉错位（ACDMPV），腺泡发育不良（AcDys），先天性肺泡发育不良（CAD）， 以及其他未指明的原发性PH。我们发现，杂合子单核苷酸变异（SNV）在 间充质转录因子（TF）FOXF 1基因或涉及FOXF 1的拷贝数变异体（CNV）缺失 或其位于上游约300 kb的肺特异性增强子负责80-90%的患者中的ACDMPV。我们 报道，该增强子也上调FENDRR顺式lncRNA定位在FOXF 1附近。有趣的是， 与SNV不同，CNV缺失几乎只发生在母体16号染色体上。最近，我们和其他 证明了另一种间充质TF，TBX 4和旁分泌信号中变体的致病作用， 在超过60%的患有AcDys、CAD和其他原发性PH的婴儿中， TBX 4-FGF 10信号传导在LLDD发病机制中也具有重要意义。重要的是，FOXF 1和TBX 4变体 也与更常见的特发性或家族性儿童PAH相关。有趣的是，我们发现 非编码SNV在FOXF 1和TBX 4增强子中的统计学显著富集， LLDD的变量表示。此外，我们的ChIP-seq和RNA-seq研究暗示了 SHH-FOXF 1和TBX 4-FGF 10信号通路，涉及鲜为人知的肺特异性内皮细胞 跨膜蛋白TMEM 100。我们假设（i）在调控区域内的非编码SNV， 肺发育基因可以显著改变（减轻或加重）LLDD和PAH表型，（ii） 编码和非编码变体之间的相互作用可以解释观察到的复杂的化合物遗传 在LLDD和PAH家族中，以及（iii）SHH-FOXF 1和TBX 4-FGF 10信号传导途径的相互作用， 涉及TMEM 100的细胞因子是正常的人肺发育所必需的。使用人类肺部标本和细胞系 和小鼠模型，我们将确定和分析FOXF 1的非编码调控元件在患者 ACDMPV和/或PAH（Aim 1）以及AcDys、CAD、其他PH和/或PAH患者中TBX 4和FGF 10的水平 PAH（目标2）。在目标3中，我们将破译SHH-FOXF 1和TBX 4-FGF 10上皮细胞之间的串扰， 间充质信号，涉及TMEM 100，解开复杂的复合遗传的家庭， LLDD和PAH。我们的研究将阐明人类肺部发育的遗传学及其扰动是如何发生的。 转化为LLDD和PAH的表型变异性。我们将发现新的基因变异， 这些疾病的精确诊断和预后，促进更多信息的遗传咨询， 为开发LLDD和PAH的潜在宫内治疗提供靶点。我们的数据也将有助于 更好地理解人类遗传学中的不完全遗传和可变表达现象。

Project Summary Lethal lung developmental disorders (LLDDs) are rarely diagnosed but devastating pulmonary hypoplasias (PHs), presenting with progressive neonatal hypoxia and severe pulmonary arterial hypertension (PAH). Based on histopathological appearance, LLDDs have been traditionally classified as alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), acinar dysplasia (AcDys), congenital alveolar dysplasia (CAD), and other unspecified primary PHs. We found that heterozygous single nucleotide variants (SNVs) in the mesenchymal transcription factor (TF) FOXF1 gene or copy-number variant (CNV) deletions involving FOXF1 or its lung-specific enhancer located ~ 300 kb upstream are responsible for ACDMPV in 80-90% of patients. We reported that this enhancer also up-regulates in cis lncRNA FENDRR mapping nearby FOXF1. Interestingly, unlike SNVs, CNV deletions arise almost exclusively on the maternal chromosome 16. Recently, we and others demonstrated the causative role for variants in another mesenchymal TF, TBX4, and a paracrine signaling molecule FGF10 in greater than 60% of infants with AcDys, CAD, and other primary PHs, indicating the significance also of TBX4-FGF10 signaling in pathogenesis of LLDDs. Importantly, FOXF1 and TBX4 variants have been associated also with more common idiopathic or familial childhood PAH. Interestingly, we found a statistically significant enrichment of non-coding SNVs in the FOXF1 and TBX4 enhancers in patients with variable presentation of LLDD. Moreover, our ChIP-seq and RNA-seq studies have implied interactions between the SHH-FOXF1 and TBX4-FGF10 signaling pathways, involving little-known lung-specific endothelial transmembrane protein TMEM100. We hypothesize that (i) non-coding SNVs within the regulatory regions of lung developmental genes can dramatically modify (alleviate or exacerbate) LLDD and PAH phenotypes, (ii) an interplay between the coding and non-coding variants can explain the complex compound inheritance observed in families with LLDDs and PAH, and (iii) interaction of SHH-FOXF1 and TBX4-FGF10 signaling pathways, involving TMEM100, is required for proper human lung development. Using human lung specimens and cell lines and mouse models, we will identify and analyze non-coding regulatory elements of FOXF1 in patients with ACDMPV and/or PAH (Aim 1) and those of TBX4 and FGF10 in patients with AcDys, CAD, other PHs, and/or PAH (Aim 2). In Aim 3, we will decipher the crosstalk between SHH-FOXF1 and TBX4-FGF10 epithelial- mesenchymal signaling, involving TMEM100, to untangle the complex compound inheritance in families with LLDDs and PAH. Our studies will elucidate the genetics of lung development in humans and how its perturbations translate to phenotypic variability of LLDDs and PAH. We will identify new genetic variants, allowing for more precise diagnosis and prognosis of these disorders, facilitating more informative genetic counseling, and providing targets for development of potential in utero treatments for LLDDs and PAH. Our data will also help to better understand incomplete penetrance and variable expressivity phenomena in human genetics in general.