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 ...