ABSTRACT
The respiratory endoderm develops from a small cluster of cells located on the ventral anterior foregut. This population of progenitors generates the myriad epithelial lineages required for proper lung function in adults through a complex and delicately balanced series of developmental events controlled by many critical signaling and transcription factor pathways. In the past decade, understanding of this process has grown enormously, helped in part by cell lineage fate analysis and deep sequencing of the transcriptomes of various progenitors and differentiated cell types. This review explores how these new techniques, coupled with more traditional approaches, have provided a detailed picture of development of the epithelial lineages in the lung and insight into how aberrant development can lead to lung disease.
Subject(s)
Endoderm/physiology , Gene Expression Regulation, Developmental/physiology , Lung/physiology , Morphogenesis/physiology , Animals , Cell Lineage/physiology , Humans , Organogenesis/physiologyABSTRACT
Transcription factors (TFs) are dosage-sensitive master regulators of gene expression, with haploinsufficiency frequently leading to life-threatening disease. Numerous mechanisms have evolved to tightly regulate the expression and activity of TFs at the transcriptional, translational, and posttranslational levels. A subset of long noncoding RNAs (lncRNAs) is spatially correlated with transcription factors in the genome, but the regulatory relationship between these lncRNAs and their neighboring TFs is unclear. We identified a regulatory feedback loop between the TF Foxa2 and a downstream lncRNA, Falcor (Foxa2-adjacent long noncoding RNA). Foxa2 directly represses Falcor expression by binding to its promoter, while Falcor functions in cis to positively regulate the expression of Foxa2. In the lung, loss of Falcor is sufficient to lead to chronic inflammatory changes and defective repair after airway epithelial injury. Moreover, disruption of the Falcor-Foxa2 regulatory feedback loop leads to altered cell adhesion and migration, in turn resulting in chronic peribronchial airway inflammation and goblet cell metaplasia. These data reveal that the lncRNA Falcor functions within a regulatory feedback loop to fine-tune the expression of Foxa2, maintain airway epithelial homeostasis, and promote regeneration.
Subject(s)
Epithelial Cells/metabolism , Hepatocyte Nuclear Factor 3-beta/genetics , Lung/cytology , Lung/metabolism , RNA, Long Noncoding/genetics , Animals , Cell Adhesion , Cell Line , Cell Movement , Female , Gene Expression Regulation , Hepatocyte Nuclear Factor 3-beta/metabolism , Homeostasis , Humans , Male , Mice , Promoter Regions, Genetic , Regeneration , Transcription, GeneticABSTRACT
We report a case of a twin-twin transfusion syndrome (TTTS) recipient who, after successful fetoscopic surgery, developed a large pericardial effusion and calcifications of the aorta and main pulmonary artery. The donor fetus never had cardiac strain and never developed cardiac calcifications. A heterozygous likely pathogenic variant in ABCC6 (c.2018T > C, p.Leu673Pro) was identified in the recipient twin. While TTTS recipient twins are at risk of arterial calcifications and right heart failure secondary to the disease, calcifications of the great vessels are also observed in generalized arterial calcification of infancy, a Mendelian genetic disorder with associated biallelic pathogenic variations in ABCC6 or ENPP1, which can result in significant pediatric morbidity or mortality. The recipient twin in this case had some degree of cardiac strain prior to TTTS surgery; however, the progressive calcification of the aorta and pulmonary trunk occurred weeks after TTTS resolution. This case raises the possibility of a gene-environment interaction and emphasizes the need for genetic evaluation in the setting of TTTS and calcifications.
Subject(s)
Fetofetal Transfusion , Female , Humans , Pregnancy , Fetofetal Transfusion/complications , Fetoscopy , Fetus/pathology , Gene-Environment Interaction , Multidrug Resistance-Associated Proteins , TwinsABSTRACT
OBJECTIVES: To identify factors associated with prenatal, perinatal, and postnatal outcomes, and determine medical care use for fetuses and infants with trisomy 13 (T13) and trisomy 18 (T18). STUDY DESIGN: This population-based retrospective cohort study included all prenatal and postnatal diagnoses of T13 or T18 in the greater Cincinnati area from January 1, 2012, to December 31, 2018. Overall survival, survival to hospital discharge, medical management, and maternal, fetal, and neonatal characteristics are analyzed. RESULTS: There were 124 pregnancies (125 fetuses) that were identified, which resulted in 72 liveborn infants. Male fetal sex and hydrops were associated with a higher rate of spontaneous loss. The median length of survival was 7 and 29 days, for infants with T13 and T18, respectively. Of the 27 infants alive at 1 month of age, 13 (48%) were alive at 1 year of age. Only trisomy type (T13), goals of care (comfort care), and extremely low birthweight were associated with a shorter length of survival. A high degree of variability existed in the use of medical services, with 28% of infants undergoing at least 1 surgical procedure and some children requiring repeated (≤29) or prolonged (>1 year) hospitalizations. CONCLUSIONS: Although many infants with T13 or T18 did not survive past the first week of life, nearly 20% lived for more than 1 year with varying degrees of medical support. The length of survival for an infant cannot be easily predicted, and surviving infants have high health care use throughout their lifespans.
Subject(s)
Fetus , Trisomy , Adolescent , Child , Chromosomes, Human, Pair 18/genetics , Female , Humans , Infant , Infant, Newborn , Male , Pregnancy , Prenatal Diagnosis/methods , Retrospective Studies , Trisomy/diagnosis , Trisomy 13 Syndrome/diagnosis , Trisomy 18 Syndrome/diagnosisABSTRACT
Long noncoding RNAs (lncRNAs) are thought to play important roles in regulating gene transcription, but few have well-defined expression patterns or known biological functions during mammalian development. Using a conservative pipeline to identify lncRNAs that have important biological functions, we identified 363 lncRNAs in the lung and foregut endoderm. Importantly, we show that these lncRNAs are spatially correlated with transcription factors across the genome. In-depth expression analyses of lncRNAs with genomic loci adjacent to the critical transcription factors Nkx2.1, Gata6, Foxa2 (forkhead box a2), and Foxf1 mimic the expression patterns of their protein-coding neighbor. Loss-of-function analysis demonstrates that two lncRNAs, LL18/NANCI (Nkx2.1-associated noncoding intergenic RNA) and LL34, play distinct roles in endoderm development by controlling expression of critical developmental transcription factors and pathways, including retinoic acid signaling. In particular, we show that LL18/NANCI acts upstream of Nkx2.1 and downstream from Wnt signaling to regulate lung endoderm gene expression. These studies reveal that lncRNAs play an important role in foregut and lung endoderm development by regulating multiple aspects of gene transcription, often through regulation of transcription factor expression.
Subject(s)
Gene Expression Regulation, Developmental , Lung/growth & development , RNA, Long Noncoding/metabolism , Transcription Factors/metabolism , Animals , Cell Differentiation/genetics , Cell Line , Endoderm/cytology , Endoderm/metabolism , Gene Expression Profiling , Gene Knockdown Techniques , Genome , HEK293 Cells , Humans , Lung/embryology , Mice , Nuclear Proteins/genetics , RNA, Long Noncoding/genetics , Thyroid Nuclear Factor 1 , Transcription Factors/genetics , Wnt Signaling PathwayABSTRACT
PURPOSE: Previous studies have reported that prenatal exome sequencing (pES) can detect monogenic diseases in fetuses with congenital anomalies with diagnostic yields ranging from 6% to 81%, but there are few reports of its clinical utility. METHODS: We conducted a retrospective chart review of patients who had pES to determine whether results led to clinical management changes. RESULTS: Of 20 patients, 8 (40%) received a definitive diagnosis. Seven patients (35%) had medical management changes based on the pES results, including alterations to their delivery plan and neonatal management (such as use of targeted medications, subspecialty referrals, additional imaging and/or procedures). All patients who received a definitive diagnosis and one who received a likely pathogenic variant (n = 9; 45%) received specific counseling about recurrence risk and the medical/developmental prognosis for the baby. In five (25%) cases, the result facilitated a diagnosis in parents and/or siblings. CONCLUSION: pES results can have significant impacts on clinical management, some of which would not be possible if testing is deferred until after birth. To maximize the clinical utility, pES should be prioritized in cases where multiple care options are available and the imaging findings alone are not sufficient to guide parental decision-making, or where postnatal testing will not be feasible.
Subject(s)
Exome , Prenatal Diagnosis , Exome/genetics , Female , Fetus , Humans , Infant, Newborn , Pregnancy , Retrospective Studies , Exome SequencingABSTRACT
OBJECTIVE: To investigate the prevalence of genetic disease and its economic impact in a level IV neonatal intensive care unit (NICU) by identifying and describing diseases diagnosed, genetic testing methodologies used, timing of diagnosis, length of NICU stay, and charges for NICU care. STUDY DESIGN: A retrospective chart review of patients admitted to a level IV NICU from 2013 to 2014 (n = 1327) was undertaken and data collected up to 2 years of age from the electronic medical record. RESULTS: In total, 117 patients (9%) received 120 genetic diagnoses using a variety of methodologies. A significant minority of diagnoses, 36%, were made after NICU discharge and 41% were made after 28 days of age. Patients receiving a genetic diagnosis had significantly longer mean lengths of stay (46 days vs 29.1 days; P < .01) and costlier mean charges ($598 712 vs $352 102; P < .01) for their NICU care. The NICU stay charge difference to care for a newborn with a genetic condition was on average $246â610 in excess of that for a patient without a genetic diagnosis, resulting in more than $28â000â000 in excess charges to care for all patients with genetic conditions in a single NICU over a 2-year period. CONCLUSIONS: Given the high prevalence of genetic disease in this population and the documented higher cost of care, shortening the time to diagnosis and targeting therapeutic interventions for this population could make a significant impact on neonatal care in level IV NICUs.
Subject(s)
Genetic Diseases, Inborn/economics , Genetic Diseases, Inborn/genetics , Genetic Testing/economics , Genetic Testing/methods , Intensive Care Units, Neonatal , Intensive Care, Neonatal/economics , DNA Methylation , Electronic Health Records , Exome , Female , Genetic Diseases, Inborn/diagnosis , Humans , In Situ Hybridization, Fluorescence , Infant , Infant Mortality , Infant, Newborn , Length of Stay , Male , Oligonucleotide Array Sequence Analysis , Patient Discharge , Prevalence , Retrospective Studies , Sequence Analysis, DNAABSTRACT
RATIONALE: Disruption of normal pulmonary development is a leading cause of morbidity and mortality in infants. Congenital lung malformations are a unique model to study the molecular pathogenesis of isolated structural birth defects, as they are often surgically resected. OBJECTIVES: To provide insight into the molecular pathogenesis of congenital lung malformations through analysis of cell-type and gene expression changes in these lesions. METHODS: Clinical data, and lung tissue for DNA, RNA, and histology, were obtained from 58 infants undergoing surgical resection of a congenital lung lesion. Transcriptome-wide gene expression analysis was performed on paired affected and unaffected samples from a subset of infants (n = 14). A three-dimensional organoid culture model was used to assess isolated congenital lung malformation epithelium (n = 3). MEASUREMENTS AND MAIN RESULTS: Congenital lung lesions express higher levels of airway epithelial related genes, and dysregulated expression of genes related to the Ras and PI3K-AKT-mTOR (phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin) signaling pathways. Immunofluorescence confirmed differentiated airway epithelial cell types throughout all major subtypes of congenital lung lesions, and three-dimensional cell culture demonstrated a cell-autonomous defect in the epithelium of these lesions. CONCLUSIONS: This study provides the first comprehensive analysis of the congenital lung malformation transcriptome and suggests that disruptions in Ras or PI3K-AKT-mTOR signaling may contribute to the pathology through an epithelial cell-autonomous defect.
Subject(s)
Cystic Adenomatoid Malformation of Lung, Congenital/genetics , Cystic Adenomatoid Malformation of Lung, Congenital/surgery , Genetic Predisposition to Disease , Lung/physiopathology , Respiratory System Abnormalities/genetics , Respiratory System Abnormalities/surgery , Female , Humans , Infant , Infant, Newborn , Male , Pennsylvania , PhenotypeABSTRACT
Background Association studies of various gene variants in neonatal sepsis show conflicting results. Objective We performed a systematic review of candidate gene association studies in neonatal sepsis to provide pooled estimates of risk for selected gene variants. Methods We performed a search using MeSH terms "infection," "sepsis," "infant," "genetic variation," "polymorphism," and "genetic association studies." We included studies evaluating associations between neonatal sepsis and genetic variants (2000-2015). We excluded case reports/series, commentaries, narrative reviews, and nonhuman research. We assessed quality of studies using STREGA guidelines. Following estimation of odds ratios (ORs), data were pooled using random effects models. Results Twenty eight of 1,404 identified studies were included. Meta-analyses were performed for interleukin (IL)-6, tumor necrosis factor-α (TNF-α), and IL-10 as these gene variants were tested in multiple studies. TNF-α 308GG genotype demonstrated trends toward increased sepsis risk in the primary analysis of culture-proven sepsis (OR 1.18, 95% confidence interval [CI] 0.97-1.44). IL-10 1082GG genotype was associated with lower sepsis odds in very low-birth-weight (VLBW) infants (OR 0.51, 95% CI 0.29-0.91). Conclusion We uncovered an association between IL-10 1082 gene variation and sepsis in VLBW infants but did not identify associations between neonatal sepsis and TNF-α 308 or IL-6 gene variation. Larger cohort replication studies are required to validate these findings.
Subject(s)
Infant, Very Low Birth Weight , Interleukin-10/genetics , Interleukin-6/genetics , Neonatal Sepsis/genetics , Tumor Necrosis Factor-alpha/genetics , Genetic Association Studies , Genetic Predisposition to Disease , Humans , Infant, Newborn , Polymorphism, Single Nucleotide , Risk FactorsABSTRACT
BACKGROUND: Opitz G/BBB syndrome is a heterogeneous disorder characterised by variable expression of midline defects including cleft lip and palate, hypertelorism, laryngealtracheoesophageal anomalies, congenital heart defects, and hypospadias. The X-linked form of the condition has been associated with mutations in the MID1 gene on Xp22. The autosomal dominant form has been linked to chromosome 22q11.2, although the causative gene has yet to be elucidated. METHODS AND RESULTS: In this study, we performed whole exome sequencing on DNA samples from a three-generation family with characteristics of Opitz G/BBB syndrome with negative MID1 sequencing. We identified a heterozygous missense mutation c.1189A>C (p.Thr397Pro) in SPECC1L, located at chromosome 22q11.23. Mutation screening of an additional 19 patients with features of autosomal dominant Opitz G/BBB syndrome identified a c.3247G>A (p.Gly1083Ser) mutation segregating with the phenotype in another three-generation family. CONCLUSIONS: Previously, SPECC1L was shown to be required for proper facial morphogenesis with disruptions identified in two patients with oblique facial clefts. Collectively, these data demonstrate that SPECC1L mutations can cause syndromic forms of facial clefting including some cases of autosomal dominant Opitz G/BBB syndrome and support the original linkage to chromosome 22q11.2.
Subject(s)
Calcium-Binding Proteins/chemistry , Esophagus/abnormalities , Genes, Dominant , Genetic Predisposition to Disease , Hypertelorism/genetics , Hypospadias/genetics , Microfilament Proteins/chemistry , Mutation/genetics , Phosphoproteins/chemistry , Phosphoproteins/genetics , Adult , Base Sequence , DNA Mutational Analysis , Exons/genetics , Family , Female , Genetic Testing , Humans , Infant , Male , Microtubule Proteins/genetics , Molecular Sequence Data , Nuclear Proteins/genetics , Pedigree , Phenotype , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Transcription Factors/genetics , Ubiquitin-Protein Ligases , CalponinsABSTRACT
Discordance between clinical phenotype and genotype has multiple causes, including mosaicism. Phenotypes can be modified due to tissue distribution, or the presence of multiple abnormal cell lines with different genomic contributions. We have studied a 20-month-old female whose main phenotypes were failure to thrive, developmental delay, and patchy skin pigmentation. Initial chromosome and SNP microarray analysis of her blood revealed a non-mosaic â¼24 Mb duplication of 15q25.1q26.3 resulting from the unbalanced translocation of terminal 15q to the short arm of chromosome 15. The most common feature associated with distal trisomy 15q is prenatal and postnatal overgrowth, which was not consistent with this patient's phenotype. The phenotypic discordance, in combination with the patchy skin pigmentation, suggested the presence of mosaicism. Further analysis of skin biopsies from both hyper- and hypopigmented regions confirmed the presence of an additional cell line with the short arm of chromosome X deleted and replaced by the entire long arm of chromosome 15. The Xp deletion, consistent with a variant Turner Syndrome diagnosis, better explained the patient's phenotype. Parental studies revealed that the alterations in both cell lines were de novo and the duplicated distal 15q and the deleted Xp were from different parental origins, suggesting a mitotic event. The possible mechanism for the occurrence of two mutually exclusive structural rearrangements with both involving the long arm of chromosome 15 is discussed.
Subject(s)
Abnormalities, Multiple/genetics , Chromosome Deletion , Chromosome Disorders/genetics , Chromosomes, Human, Pair 15/genetics , Chromosomes, Human, X/genetics , Gene Duplication , Mosaicism/embryology , Trisomy/genetics , Abnormalities, Multiple/diagnosis , Chromosome Disorders/diagnosis , Comparative Genomic Hybridization , Female , Genotype , Humans , In Situ Hybridization, Fluorescence , Infant , Phenotype , Prenatal Diagnosis , Prognosis , Trisomy/diagnosisABSTRACT
Loss of function mutations in FREM1 have been demonstrated in Manitoba-oculo-tricho-anal (MOTA) syndrome and Bifid Nose Renal Agenesis and Anorectal malformations (BNAR) syndrome, but the wider phenotypic spectrum that is associated with FREM1 mutations remains to be defined. We screened three probands with phenotypic features of MOTA syndrome. In one severely affected infant who was diagnosed with MOTA syndrome because of bilateral eyelid colobomas, a bifid nasal tip, hydrometrocolpos and vaginal atresia, we found two nonsense mutations that likely result in complete loss of FREM1 function. This infant also had renal dysplasia, a finding more consistent with BNAR syndrome. Another male who was homozygous for a novel stop mutation had an extensive eyelid colobomas, corneopalpebral synechiae, and unilateral renal agenesis. A third male child diagnosed with MOTA syndrome because of corneopalpebral synechiae and eyelid colobomas had a homozygous splice site mutation in FREM1. These cases illustrate that disruption of the FREM1 gene can produce a spectrum of clinical manifestations encompassing the previously described MOTA and BNAR syndromes, and that features of both syndromes may be seen in the same individual. The phenotype of FREM1-related disorders is thus more pleiotropic than for MOTA and BNAR syndrome alone and more closely resembles the widespread clinical involvement seen with Fraser syndrome. Moreover, our first case demonstrates that vaginal atresia may be a feature of FREM1-related disorders.
Subject(s)
Abnormalities, Multiple/diagnosis , Coloboma/diagnosis , Hypertelorism/diagnosis , Kidney Diseases/diagnosis , Receptors, Interleukin/genetics , Rectal Diseases/diagnosis , Abnormalities, Multiple/genetics , Anal Canal/abnormalities , Coloboma/genetics , Consanguinity , Fatal Outcome , Female , Genetic Association Studies , Humans , Hypertelorism/genetics , Infant , Infant, Newborn , Kidney Diseases/genetics , Male , Molecular Diagnostic Techniques , Mutation , Nose/abnormalities , Phenotype , Rectal Diseases/genetics , Sequence Analysis, DNAABSTRACT
Here we describe three subjects with mosaic genome-wide paternal uniparental isodisomy (GWpUPD) each of whom presented initially with overgrowth, hemihyperplasia (HH), and hyperinsulinism (HI). Due to the severity of findings and the presence of additional features, SNP array testing was performed, which demonstrated mosaic GWpUPD. Comparing these individuals to 10 other live-born subjects reported in the literature, the predominant phenotype is that of pUPD11 and notable for a very high incidence of tumor development. Our subjects developed non-metastatic tumors of the adrenal gland, kidney, and/or liver. All three subjects had pancreatic hyperplasia resulting in HI. Notably, our subjects to date display minimal features of other diseases associated with paternal UPD loci. Both children who survived the neonatal period have displayed near-normal cognitive development, likely due to a favorable tissue distribution of the mosaicism. To understand the range of UPD mosaicism levels, we studied multiple tissues using SNP array analysis and detected levels of 5-95%, roughly correlating with the extent of tissue involvement. Given the rapidity of tumor growth and the difficulty distinguishing malignant and benign tumors in these GWpUPD subjects, we have utilized increased frequency of ultrasound (US) and alpha-fetoprotein (AFP) screening in the first years of life. Because of a later age of onset of additional tumors, continued tumor surveillance into adolescence may need to be considered in these rare patients.
Subject(s)
Chromosomes, Human, Pair 11/genetics , Genome, Human , Hyperbilirubinemia, Hereditary/genetics , Hyperinsulinism/genetics , Hyperplasia/genetics , Mosaicism , Neoplasms/genetics , Uniparental Disomy/genetics , Adult , Cells, Cultured , Child, Preschool , Chromosome Aberrations , Comparative Genomic Hybridization , Female , Genotype , Humans , Hyperbilirubinemia, Hereditary/pathology , Hyperinsulinism/pathology , Hyperplasia/pathology , Infant , Magnetic Resonance Imaging , Neoplasms/pathology , Phenotype , Polymorphism, Single Nucleotide/genetics , Uniparental Disomy/pathology , alpha-Fetoproteins/metabolismABSTRACT
OBJECTIVE: The authors of this study aimed to evaluate the use of polysomnography (PSG) in children with Down syndrome (DS) between ages 0 and 7 years, to assess the prevalence and severity of obstructive sleep apnea (OSA) and associated comorbidities, and to describe interventions used for OSA. METHODS: A retrospective cohort study was performed at Cincinnati Children's Hospital Medical Center for children with DS born between 2013 and 2019. Data were extracted from the electronic medical record, including demographics, age at PSG, PSG results, and interventions after an abnormal PSG. Statistical analysis included unadjusted bivariate association testing and multivariable logistic regression modeling to investigate associations with OSA severity. RESULTS: Among 397 patients in the cohort, 59% (n = 235) had a documented PSG and 94% (n = 221) had an abnormal study with 60% (n = 141) demonstrating moderate or severe OSA. There was an inverse relationship between age and OSA severity (P < .001). In a multiple regression model, OSA severity was associated with increased rates of failure to thrive (P < .01), aspiration (P = .02), and laryngomalacia (P < .01). After medical or surgical intervention, 73% of patients experienced the resolution of OSA or an improvement in OSA severity. CONCLUSION: In this study of pediatric patients with DS, OSA was identified most frequently in the first year of life. In addition, to prompt evaluation of symptomatic infants, our data support earlier PSG screening for patients requiring neonatal ICU care and those with feeding difficulties, airway abnormalities, and/or pulmonary hypertension given their increased risk for severe OSA.
Subject(s)
Down Syndrome , Sleep Apnea, Obstructive , Infant , Infant, Newborn , Child , Humans , Child, Preschool , Retrospective Studies , Down Syndrome/complications , Down Syndrome/epidemiology , Sleep Apnea, Obstructive/complications , Sleep Apnea, Obstructive/diagnosis , Sleep Apnea, Obstructive/epidemiology , Polysomnography/adverse effects , ComorbidityABSTRACT
Lung epithelial regeneration after acute injury requires coordination cellular coordination to pattern the morphologically complex alveolar gas exchange surface. During adult lung regeneration, Wnt-responsive alveolar epithelial progenitor (AEP) cells, a subset of alveolar type 2 (AT2) cells, proliferate and transition to alveolar type 1 (AT1) cells. Here, we report a refined primary murine alveolar organoid, which recapitulates critical aspects of in vivo regeneration. Paired scRNAseq and scATACseq followed by transcriptional regulatory network (TRN) analysis identified two AT1 transition states driven by distinct regulatory networks controlled in part by differential activity of Nkx2-1. Genetic ablation of Nkx2-1 in AEP-derived organoids was sufficient to cause transition to a proliferative stressed Krt8+ state, and AEP-specific deletion of Nkx2-1 in adult mice led to rapid loss of progenitor state and uncontrolled growth of Krt8+ cells. Together, these data implicate dynamic epigenetic maintenance via Nkx2-1 as central to the control of facultative progenitor activity in AEPs.
Subject(s)
Epigenomics , Lung , Animals , Mice , Cell Differentiation , Epithelial Cells , Homeostasis , Stem CellsABSTRACT
Differential chromatin accessibility accompanies and mediates transcriptional control of diverse cell fates and their differentiation during embryogenesis. While the critical role of NKX2-1 and its transcriptional targets in lung morphogenesis and pulmonary epithelial cell differentiation is increasingly known, mechanisms by which chromatin accessibility alters the epigenetic landscape and how NKX2-1 interacts with other co-activators required for alveolar epithelial cell differentiation and function are not well understood. Here, we demonstrate that the paired domain zinc finger transcriptional regulators PRDM3 and PRDM16 regulate chromatin accessibility to mediate cell differentiation decisions during lung morphogenesis. Combined deletion of Prdm3 and Prdm16 in early lung endoderm caused perinatal lethality due to respiratory failure from loss of AT2 cell function. Prdm3/16 deletion led to the accumulation of partially differentiated AT1 cells and loss of AT2 cells. Combination of single cell RNA-seq, bulk ATAC-seq, and CUT&RUN demonstrated that PRDM3 and PRDM16 enhanced chromatin accessibility at NKX2-1 transcriptional targets in peripheral epithelial cells, all three factors binding together at a multitude of cell-type specific cis-active DNA elements. Network analysis demonstrated that PRDM3/16 regulated genes critical for perinatal AT2 cell differentiation, surfactant homeostasis, and innate host defense. Lineage specific deletion of PRDM3/16 in AT2 cells led to lineage infidelity, with PRDM3/16 null cells acquiring partial AT1 fate. Together, these data demonstrate that NKX2-1-dependent regulation of alveolar epithelial cell differentiation is mediated by epigenomic modulation via PRDM3/16.
ABSTRACT
OBJECTIVE: To determine detection rates of genetic disease in a level IV neonatal intensive care unit (NICU) and cost of care. STUDY DESIGN: We divided 2703 neonates, admitted between 2013 and 2016 to a level IV NICU, into two epochs and determined how genetic testing utilization, genetic diagnoses identified, and cost of NICU care changed over time. RESULT: The increasing use of multi-gene panels 104 vs 184 (P = 0.02) and whole exome sequencing (WES) 9 vs 28 (P = 0.03) improved detection of genetic disease, 9% vs 12% (P < 0.01). Individuals with genetic diagnoses had higher mean NICU charges, $723,422 vs $417,013 (P < 0.01) secondary to longer lengths of stay, not genetic services. CONCLUSION: The increased utilization of broad genetic testing improved the detection of genetic disease but contributed minimally to the cost of care while bolstering understanding of the patient's condition and prognosis.
Subject(s)
Genetic Testing , Intensive Care Units, Neonatal , Hospitalization , Humans , Infant, Newborn , Exome SequencingABSTRACT
The tips of the developing respiratory buds are home to important progenitor cells marked by the expression of SOX9 and ID2. Early in embryonic development (prior to E13.5), SOX9+progenitors are multipotent, generating both airway and alveolar epithelium, but are selective progenitors of alveolar epithelial cells later in development. Transcription factors, including Sox9, Etv5, Irx, Mycn, and Foxp1/2 interact in complex gene regulatory networks to control proliferation and differentiation of SOX9+progenitors. Molecular mechanisms by which these transcription factors and other signaling pathways control chromatin state to establish and maintain cell-type identity are not well-defined. Herein, we analyze paired gene expression (RNA-Seq) and chromatin accessibility (ATAC-Seq) data from SOX9+ epithelial progenitor cells (EPCs) during embryonic development in Mus musculus. Widespread changes in chromatin accessibility were observed between E11.5 and E16.5, particularly at distal cis-regulatory elements (e.g. enhancers). Gene regulatory network (GRN) inference identified a common SOX9+ progenitor GRN, implicating phosphoinositide 3-kinase (PI3K) signaling in the developmental regulation of SOX9+ progenitor cells. Consistent with this model, conditional ablation of PI3K signaling in the developing lung epithelium in mouse resulted in an expansion of the SOX9+ EPC population and impaired airway epithelial cell differentiation. These data demonstrate that PI3K signaling is required for epithelial patterning during lung organogenesis, and emphasize the combinatorial power of paired RNA and ATAC seq in defining regulatory networks in development.
Studying how lungs develop has helped us understand and treat often-devastating lung diseases. This includes diseases like cystic fibrosis which result from spelling mistakes known as mutations in a person's genetic code. However, not all lung diseases involve mutations. Many other diseases, in both adults and children, are caused by genes failing to switch on or off at some point during lung development. DNA is surrounded by various proteins which package it into a compressed structure known as chromatin. Cells can control which genes are turned on or off by modifying how tightly packed parts of the genetic code are within chromatin. Changes in chromatin accessibility, also known as 'epigenetic' changes, are a normal part of development, and guide cells towards specific jobs or identities as an organ matures. However, how this happens in the developing lung is poorly understood. Here, Khattar, Fernandes et al. set out to determine how chromatin accessibility shapes development of the tissue lining the lungs, focusing on a group of progenitor cells which produce the protein SOX9. These cells are initially found at the tips of the early lung, where they go on to develop into the cells that line the whole of the mature organ. Initial experiments used large-scale genetic techniques to measure gene activity and chromatin accessibility simultaneously in progenitor cells extracted from the lungs of mice. Khattar, Fernandes et al. were then able to predict the signaling pathways that shape the lung lining based on which genes were surrounded by unpacked chromatin, and determine the proteins responsible for these epigenetic changes. This included the signaling pathway Phosphatidylinositol 3 kinase (PI3K) which is involved in a number of cellular processes. Additional experiments in mice confirmed that the PI3K pathway became active very early in lung development and remained so until adulthood. In contrast, mice lacking a gene that codes for a key part of the PI3K pathway had defective lungs which failed to develop a proper lining. The data generated in this study will provide an important resource for future studies investigating how epigenetic changes drive normal lung development. Khattar, Fernandes et al. hope that this knowledge will help researchers to better understand the cause of human lung diseases, and identify already available 'epigenetic drugs' which could be repurposed to treat them.
Subject(s)
Gene Regulatory Networks , Phosphatidylinositol 3-Kinases , Animals , Cell Differentiation/genetics , Chromatin , Female , Gene Expression Regulation, Developmental , Lung , Mice , Phosphatidylinositol 3-Kinase/genetics , Phosphatidylinositol 3-Kinases/genetics , PregnancyABSTRACT
Accurate estimate of fetal maturity could provide individualized guidance for delivery of complicated pregnancies. However, current methods are invasive, have low accuracy, and are limited to fetal lung maturation. To identify diagnostic gestational biomarkers, we performed transcriptomic profiling of lung and brain, as well as cell-free RNA from amniotic fluid of preterm and term rhesus macaque fetuses. These data identify potentially new and prior-associated gestational age differences in distinct lung and neuronal cell populations when compared with existing single-cell and bulk RNA-Seq data. Comparative analyses found hundreds of genes coincidently induced in lung and amniotic fluid, along with dozens in brain and amniotic fluid. These data enable creation of computational models that accurately predict lung compliance from amniotic fluid and lung transcriptome of preterm fetuses treated with antenatal corticosteroids. Importantly, antenatal steroids induced off-target gene expression changes in the brain, impinging upon synaptic transmission and neuronal and glial maturation, as this could have long-term consequences on brain development. Cell-free RNA in amniotic fluid may provide a substrate of global fetal maturation markers for personalized management of at-risk pregnancies.