Clinical Relevance of Rapid FOXF1-Targeted Sequencing in Patients Suspected of Alveolar Capillary Dysplasia With Misalignment of Pulmonary Veins.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal congenital lung disorder that presents shortly after birth with respiratory failure and therapy-resistant pulmonary hypertension. It is associated with heterozygous point mutations and genomic deletions that involve the FOXF1 gene or its upstream regulatory region. Patients are unresponsive to the intensive treatment regimens and suffer unnecessarily because ACDMPV is not always timely recognized and histologic diagnosis is invasive and time consuming. Here, we demonstrate the usefulness of a noninvasive, fast genetic test for FOXF1 variants that we previously developed to rapidly diagnose ACDMPV and reduce the time of hospitalization.

Early prenatal diagnosis of alveolar capillary dysplasia with misalignment of pulmonary veins due to a 16q24.1 deletion.

First trimester ultrasound screening is an essential fetal examination performed generally at 11-13 weeks of gestation (WG). However, it does not allow for an accurate description of all fetal organs, partly due to their development in progress. Meanwhile, increased nuchal translucency (INT) is a widely used marker known to be associated with chromosomal deleterious rearrangements. We report on a 14 WG fetus with an association of INT and univentricular congenital heart malformation (CHM) leading to chorionic villous sampling (CVS). Cytogenetic investigations performed using array-Comparative Genomic Hybridization (CGH) and fluorescence in situ hybridization (FISH) demonstrated a 1.17 Mb deletion in 16q24.1 encompassing FOXF1 arisen de novo on maternal inherited chromosome. Fetopathological study confirmed CHM with hypoplastic left heart syndrome (HLHS) associating aortic atresia, mitral stenosis, and left ventricular hypoplasia and revealed in addition specific lung lesions corresponding to alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). This is so far the first case of first trimester prenatal diagnosis of ACDMPV due to the deletion of FOXF1 gene. An interpretation of the complex genomic data generated by ultrasound markers is facilitated considerably by the genotype-phenotype correlations on fetopathological examination.

The S52F FOXF1 Mutation Inhibits STAT3 Signaling and Causes Alveolar Capillary Dysplasia.

Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal congenital disorder causing respiratory failure and pulmonary hypertension shortly after birth. There are no effective treatments for ACDMPV other than lung transplant, and new therapeutic approaches are urgently needed. Although ACDMPV is linked to mutations in the FOXF1 gene, molecular mechanisms through which FOXF1 mutations cause ACDMPV are unknown.Objectives: To identify molecular mechanisms by which S52F FOXF1 mutations cause ACDMPV.Methods: We generated a clinically relevant mouse model of ACDMPV by introducing the S52F FOXF1 mutation into the mouse Foxf1 gene locus using CRISPR/Cas9 technology. Immunohistochemistry, whole-lung imaging, and biochemical methods were used to examine vasculature in Foxf1WT/S52F lungs and identify molecular mechanisms regulated by FOXF1.Measurements and Main Results: FOXF1 mutations were identified in 28 subjects with ACDMPV. Foxf1WT/S52F knock-in mice recapitulated histopathologic findings in ACDMPV infants. The S52F FOXF1 mutation disrupted STAT3-FOXF1 protein-protein interactions and inhibited transcription of Stat3, a critical transcriptional regulator of angiogenesis. STAT3 signaling and endothelial proliferation were reduced in Foxf1WT/S52F mice and human ACDMPV lungs. S52F FOXF1 mutant protein did not bind chromatin and was transcriptionally inactive. Furthermore, we have developed a novel formulation of highly efficient nanoparticles and demonstrated that nanoparticle delivery of STAT3 cDNA into the neonatal circulation restored endothelial proliferation and stimulated lung angiogenesis in Foxf1WT/S52F mice.Conclusions: FOXF1 acts through STAT3 to stimulate neonatal lung angiogenesis. Nanoparticle delivery of STAT3 is a promising strategy to treat ACDMPV associated with decreased STAT3 signaling.

Genomic and Epigenetic Complexity of the FOXF1 Locus in 16q24.1: Implications for Development and Disease.

The FOXF1 (Forkhead box F1) gene, located on chromosome 16q24.1 encodes a member of the FOX family of transcription factors characterized by a distinct forkhead DNA binding domain. FOXF1 plays an important role in epithelium-mesenchyme signaling, as a downstream target of Sonic hedgehog pathway. Heterozygous point mutations and genomic deletions involving FOXF1 have been reported in newborns with a lethal lung developmental disorder, Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV). In addition, genomic deletions upstream to FOXF1 identified in ACDMPV patients have revealed that FOXF1 expression is tightly regulated by distal tissue-specific enhancers. Interestingly, FOXF1 has been found to be incompletely paternally imprinted in human lungs; characterized genomic deletions arose de novo exclusively on maternal chromosome 16, with most of them being Alu-Alu mediated. Regulation of FOXF1 expression likely utilizes a combination of chromosomal looping, differential methylation of an upstream CpG island overlapping GLI transcription factor binding sites, and the function of lung-specific long non-coding RNAs (lncRNAs). FOXF1 knock-out mouse models demonstrated its critical role in mesoderm differentiation and in the development of pulmonary vasculature. Additionally, epigenetic inactivation of FOXF1 has been reported in breast and colorectal cancers, whereas overexpression of FOXF1 has been associated with a number of other human cancers, e.g. medulloblastoma and rhabdomyosarcoma. Constitutional duplications of FOXF1 have recently been reported in congenital intestinal malformations. Thus, understanding the genomic and epigenetic complexity at the FOXF1 locus will improve diagnosis, prognosis, and treatment of ACDMPV and other human disorders associated with FOXF1 alterations.

Two deletions overlapping a distant FOXF1 enhancer unravel the role of lncRNA LINC01081 in etiology of alveolar capillary dysplasia with misalignment of pulmonary veins.

Position effects due to disruption of distant cis-regulatory regions have been reported for over 40 human gene loci; however, the underlying mechanisms of long-range gene regulation remain largely unknown. We report on two patients with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) caused by overlapping genomic deletions that included a distant FOXF1 transcriptional enhancer mapping 0.3 Mb upstream to FOXF1 on 16q24.1. In one patient with atypical late-onset ACDMPV, a ∼1.5 Mb deletion removed the proximal 43% of this enhancer, leaving the lung-specific long non-coding RNA (lncRNA) gene LINC01081 intact. In the second patient with severe neonatal-onset ACDMPV, an overlapping ∼194 kb deletion disrupted LINC01081. Both deletions arose de novo on maternal copy of the chromosome 16, supporting the notion that FOXF1 is paternally imprinted in the human lungs. RNAi-mediated knock-down of LINC01081 in normal fetal lung fibroblasts showed that this lncRNA positively regulates FOXF1 transcript level, further indicating that decrease in LINC01081 expression can contribute to development of ACDMPV.

Demonstration of Safety in Wild Type Mice of npFOXF1, a Novel Nanoparticle-Based Gene Therapy for Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins.

Introduction: Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins (ACDMPV) is a fatal congenital disease resulting from a pulmonary vascular endothelial deficiency of FOXF1, producing abnormal morphogenesis of alveolar capillaries, malpositioned pulmonary veins and disordered development of lung lobes. Affected neonates suffer from cyanosis, severe breathing insufficiency, pulmonary hypertension, and death typically within days to weeks after birth. Currently, no treatment exists for ACDMPV, although recent murine research in the Kalinichenko lab demonstrates nanoparticle delivery improves survival and reconstitutes normal alveolar-capillary architecture. The aim of the present study is to investigate the safety of intravenous administration of FOXF1-expressing PEI-PEG nanoparticles (npFOXF1), our pioneering treatment for ACDMPV. Methods: npFOXF1 was constructed, validated, and subsequently administered in a single dose to postnatal day 14 (P14) mice via retro-orbital injection. Biochemical, serologic, and histologic safety were monitored at postnatal day 16 (P16) and postnatal day 21 (P21). Results: With treatment we observed no lethality, and the general condition of mice revealed no obvious abnormalities. Serum chemistry, whole blood, and histologic toxicity was assayed on P16 and P21 and revealed no abnormality. Discussion: In conclusion, npFOXF1 has a very good safety profile and combined with preceding studies showing therapeutic efficacy, npFOXF1 can be considered as a good candidate therapy for ACDMPV in human neonates.

Further refinement of the differentially methylated distant lung-specific FOXF1 enhancer in a neonate with alveolar capillary dysplasia.

Heterozygous SNVs or CNV deletions involving the FOXF1 gene, or its distant enhancer, are causative for 80-90% of cases of alveolar capillary dysplasia with misalignment of pulmonary veins. Recently, we proposed bimodal structure and parental functional dimorphism of the lung-specific FOXF1 enhancer, with Unit 1 having higher activity on the paternal chr16 and Unit 2 on the maternal chr16. Here, we describe a novel unusually sized pathogenic de novo copy-number variant deletion involving a portion of the FOXF1 enhancer on maternal chr16 that implies narrowing Unit 2 to an essential ~ 9-kb segment. Using a restrictase-based assay, we found that this enhancer segment is weakly methylated at ApT adenine, with about twice the frequency of methylation on the maternal versus paternal chr16. Our data provide further insight into the FOXF1 enhancer structure and function.

A Small De Novo CNV Deletion of the Paternal Copy of FOXF1, Leaving lncRNA FENDRR Intact, Provides Insight into Their Bidirectional Promoter Region.

Pathogenic single-nucleotide variants (SNVs) and copy-number variant (CNV) deletions involving the FOXF1 transcription factor gene or CNV deletions of its distant lung-specific enhancer are responsible for alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a rarely diagnosed lethal lung developmental disorder in neonates. In contrast to SNVs within FOXF1 and CNV deletions involving only the FOXF1 enhancer, larger-sized deletions involving FOXF1 and the adjacent, oppositely oriented lncRNA gene FENDRR have additionally been associated with hypoplastic left heart syndrome and single umbilical artery (SUA). Here, in an ACDMPV infant without any congenital heart defect or SUA, we identified a small 5 kb CNV deletion that removed the paternal allele of FOXF1 and its promoter, leaving FENDRR and its promoter intact. Reporter assay in the IMR-90 fetal cell line implied that the deletion may indeed not have significantly affected FENDRR expression. It also showed a polarization of the FOXF1-FENDRR inter-promoter region consisting of its ability to increase the transcription of FENDRR but not FOXF1. Interestingly, this transcription-stimulating activity was suppressed in the presence of the FOXF1 promoter. Our data shed more light on the interactions between neighboring promoters of FOXF1-FENDRR and possibly other divergently transcribed mRNA-lncRNA gene pairs.

Unusual presentation of alveolar capillary dysplasia with misalignment of the pulmonary veins in a child with respiratory syncytial virus pneumonia: A case report.

Alveolar capillary dysplasia with misalignment of the pulmonary veins (ACDMPV) is a rare congenital diffuse lung disorder, with a fatal course during the neonatal period. We describe an 18-month-old boy who presented with respiratory syncytial virus pneumonia and pulmonary hypertensive crisis requiring extracorporeal membrane oxygenation. Exome sequencing revealed a FOXF1 frameshift variant, NM_001451.2:c.995_998delACTC, inherited from his asymptomatic mother. Genetic findings were compatible with histopathology findings from a lung biopsy. Based on the disease course, histopathology, and outcomes of this case, we believe ACDMPV should be considered a possibility in an infant presenting with hypoxemic respiratory failure, resistant pulmonary hypertension, and vasodilator-induced pulmonary edema. Genetic testing can contribute to the diagnostic process.

Haploinsufficiencies of FOXF1, FOXC2 and FOXL1 genes originated from deleted 16q24.1q24.2 fragment related with alveolar capillary dysplasia with misalignment of pulmonary veins and lymphedema-distichiasis syndrome: relationship to phenotype.

OBJECTIVE: We describe a fetus with a 2.12-Mb terminal deleted fragment in 16q associated with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) and lymphedema-distichiasis syndrome (LDS) and intend to provide a comprehensive prenatal management strategy for the fetuses with ACDMPV and LDS through reviewing other similar published studies. METHODS: The fetus presented a series of diverse structural malformations including congenital cardiovascular, genitourinary and gastro-intestinal anomalies in ultrasound at 23 + 5 weeks of gestation (GA). Amniocentesis was conducted for karyotype analysis and copy number variation sequencing (CNV-seq) after informed consent. RESULTS: The fetal karyotype was 46,XX, however the result of CNV-seq showed an approximately 2.12-Mb deletion in 16q24.1q24.2 (85220000-87340000) × 1 indicating pathogenicity. CONCLUSION: Genomic testing should be recommend as a first line diagnostic tool for suspected ACDMPV and/or LDS or other genetic syndromes for the fetuses with structural abnormalities in clinical practice.

Genome wide DNA methylation analysis of alveolar capillary dysplasia lung tissue reveals aberrant methylation of genes involved in development including the FOXF1 locus.

BACKGROUND: Alveolar capillary dysplasia with or without misalignment of the pulmonary veins (ACD/MPV) is a lethal congenital lung disorder associated with a variety of heterozygous genomic alterations in the FOXF1 gene or its 60 kb enhancer. Cases without a genomic alteration in the FOXF1 locus have been described as well. The mechanisms responsible for FOXF1 haploinsufficiency and the cause of ACD/MPV in patients without a genomic FOXF1 variant are poorly understood, complicating the search for potential therapeutic targets for ACD/MPV. To investigate the contribution of aberrant DNA methylation, genome wide methylation patterns of ACD/MPV lung tissues were compared with methylation patterns of control lung tissues using the recently developed technique Methylated DNA sequencing (MeD-seq). RESULTS: Eight ACD/MPV lung tissue samples and three control samples were sequenced and their mutual comparison resulted in identification of 319 differentially methylated regions (DMRs) genome wide, involving 115 protein coding genes. The potentially upregulated genes were significantly enriched in developmental signalling pathways, whereas potentially downregulated genes were mainly enriched in O-linked glycosylation. In patients with a large maternal deletion encompassing the 60 kb FOXF1 enhancer, DNA methylation patterns in this FOXF1 enhancer were not significantly different compared to controls. However, two hypermethylated regions were detected in the 60 kb FOXF1 enhancer of patients harbouring a FOXF1 point mutation. Lastly, a large hypermethylated region overlapping the first FOXF1 exon was found in one of the ACD/MPV patients without a known pathogenic FOXF1 variation. CONCLUSION: This is the first study providing genome wide methylation data on lung tissue of ACD/MPV patients. DNA methylation analyses in the FOXF1 locus excludes maternal imprinting of the 60 kb FOXF1 enhancer. Hypermethylation at the 60 kb FOXF1 enhancer might contribute to FOXF1 haploinsufficiency caused by heterozygous mutations in the FOXF1 coding region. Interestingly, DNA methylation analyses of patients without a genomic FOXF1 variant suggest that abnormal hypermethylation of exon 1 might play a role in some ACD/MPV in patients.

Novel parent-of-origin-specific differentially methylated loci on chromosome 16.

BACKGROUND: Congenital malformations associated with maternal uniparental disomy of chromosome 16, upd(16)mat, resemble those observed in newborns with the lethal developmental lung disease, alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Interestingly, ACDMPV-causative deletions, involving FOXF1 or its lung-specific upstream enhancer at 16q24.1, arise almost exclusively on the maternally inherited chromosome 16. Given the phenotypic similarities between upd(16)mat and ACDMPV, together with parental allelic bias in ACDMPV, we hypothesized that there may be unknown imprinted loci mapping to chromosome 16 that become functionally unmasked by chromosomal structural variants. RESULTS: To identify parent-of-origin biased DNA methylation, we performed high-resolution bisulfite sequencing of chromosome 16 on peripheral blood and cultured skin fibroblasts from individuals with maternal or paternal upd(16) as well as lung tissue from patients with ACDMPV-causative 16q24.1 deletions and a normal control. We identified 22 differentially methylated regions (DMRs) with ≥ 5 consecutive CpG methylation sites and varying tissue-specificity, including the known DMRs associated with the established imprinted gene ZNF597 and DMRs supporting maternal methylation of PRR25, thought to be paternally expressed in lymphoblastoid cells. Lastly, we found evidence of paternal methylation on 16q24.1 near LINC01082 mapping to the FOXF1 enhancer. CONCLUSIONS: Using high-resolution bisulfite sequencing to evaluate DNA methylation across chromosome 16, we found evidence for novel candidate imprinted loci on chromosome 16 that would not be evident in array-based assays and could contribute to the birth defects observed in patients with upd(16)mat or in ACDMPV.

Lethal lung hypoplasia and vascular defects in mice with conditional Foxf1 overexpression.

FOXF1 heterozygous point mutations and genomic deletions have been reported in newborns with the neonatally lethal lung developmental disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). However, no gain-of-function mutations in FOXF1 have been identified yet in any human disease conditions. To study the effects of FOXF1 overexpression in lung development, we generated a Foxf1 overexpression mouse model by knocking-in a Cre-inducible Foxf1 allele into the ROSA26 (R26) locus. The mice were phenotyped using micro-computed tomography (micro-CT), head-out plethysmography, ChIP-seq and transcriptome analyses, immunohistochemistry, and lung histopathology. Thirty-five percent of heterozygous R26-Lox-Stop-Lox (LSL)-Foxf1 embryonic day (E)15.5 embryos exhibit subcutaneous edema, hemorrhages and die perinatally when bred to Tie2-cre mice, which targets Foxf1 overexpression to endothelial and hematopoietic cells. Histopathological and micro-CT evaluations revealed that R26Foxf1; Tie2-cre embryos have immature lungs with a diminished vascular network. Neonates exhibited respiratory deficits verified by detailed plethysmography studies. ChIP-seq and transcriptome analyses in E18.5 lungs identified Sox11, Ghr, Ednrb, and Slit2 as potential downstream targets of FOXF1. Our study shows that overexpression of the highly dosage-sensitive Foxf1 impairs lung development and causes vascular abnormalities. This has important clinical implications when considering potential gene therapy approaches to treat disorders of FOXF1 abnormal dosage, such as ACDMPV.