TBX5 pathogenic variant in a patient with congenital heart defect and tracheal stenosis.

Congenital tracheal stenosis is a rare life-threatening disorder caused by narrow O-shaped tracheal ring without smooth muscle. Its underlying genetic cause has not been elucidated. We performed whole exome sequencing in a patient with congenital tracheal stenosis and congenital heart defect, and identified a de novo pathogenic TBX5 variant (NM_181486.4:c.680T>C, p.(Ile227Thr)). The Ile227Thr-TBX5 protein was predicted to have a decreased stability by in silico protein structural analyses, and was shown to have a significantly reduced activity for the NPPA promoter by luciferase assay. The results, together with the expression of mouse Tbx5 in the lung and trachea and the development of tracheal cartilage dysplasia in the lung-specific Tbx5 null mice, imply the relevance of TBX5 pathogenic variants to congenital tracheal stenosis.

IL-11 drives the phenotypic transformation of tracheal epithelial cells and fibroblasts to enhance abnormal repair after tracheal injury.

Tracheal stenosis (TS) is a multifactorial and heterogeneous disease that can easily lead to respiratory failure and even death. Interleukin-11 (IL-11) has recently received increased attention as a fibrogenic factor, but its function in TS is uncertain. This study aimed to investigate the role of IL-11 in TS regulation based on clinical samples from patients with TS and a rat model of TS produced by nylon brush scraping. Using lentiviral vectors expressing shRNA (lentivirus-shRNA) targeting the IL-11 receptor (IL-11Rα), we lowered IL-11Rα levels in the rat trachea. Histological and immunostaining methods were used to evaluate the effects of IL-11Rα knockdown on tracheal injury, molecular phenotype, and fibrosis in TS rats. We show that IL-11 was significantly elevated in circulating serum and granulation tissue in patients with TS. In vitro, TGFß1 dose-dependently stimulated IL-11 secretion from human tracheal epithelial cells (Beas-2b) and primary rat tracheal fibroblasts (PRTF). IL-11 transformed the epithelial cell phenotype to the mesenchymal cell phenotype by activating the ß-catenin pathway. Furthermore, IL-11 activated the atypical ERK signaling pathway, stimulated fibroblasts proliferation, and transformed fibroblasts into alpha-smooth muscle actin (α-SMA) positive myofibroblasts. IL-11-neutralizing antibodies (IL-11NAb) or ERK inhibitors (U0126) inhibited IL-11 activity and downregulated fibrotic responses involving TGFß/SMAD signaling. In vivo, IL-11Rα knockdown rats showed unobstructed tracheal lumen, relatively intact epithelial structure, and significantly reduced granulation tissue proliferation and collagen fiber deposition. Our findings confirm that IL-11 may be a target for future drug prevention and treatment of tracheal stenosis.

Molecular Mechanisms and Physiological Changes behind Benign Tracheal and Subglottic Stenosis in Adults.

Laryngotracheal stenosis (LTS) is a complex and heterogeneous disease whose pathogenesis remains unclear. LTS is considered to be the result of aberrant wound-healing process that leads to fibrotic scarring, originating from different aetiology. Although iatrogenic aetiology is the main cause of subglottic or tracheal stenosis, also autoimmune and infectious diseases may be involved in causing LTS. Furthermore, fibrotic obstruction in the anatomic region under the glottis can also be diagnosed without apparent aetiology after a comprehensive workup; in this case, the pathological process is called idiopathic subglottic stenosis (iSGS). So far, the laryngotracheal scar resulting from airway injury due to different diseases was considered as inert tissue requiring surgical removal to restore airway patency. However, this assumption has recently been revised by regarding the tracheal scarring process as a fibroinflammatory event due to immunological alteration, similar to other fibrotic diseases. Recent acquisitions suggest that different factors, such as growth factors, cytokines, altered fibroblast function and genetic susceptibility, can all interact in a complex way leading to aberrant and fibrotic wound healing after an insult that acts as a trigger. However, also physiological derangement due to LTS could play a role in promoting dysregulated response to laryngo-tracheal mucosal injury, through biomechanical stress and mechanotransduction activation. The aim of this narrative review is to present the state-of-the-art knowledge regarding molecular mechanisms, as well as mechanical and physio-pathological features behind LTS.

Pilot Gene Expression and Histopathologic Analysis of Tracheal Resections in Tracheobronchomalacia.

BACKGROUND: The airway structures and messenger RNA expression of genes that regulate airway inflammation and remodeling may be altered in the trachea of patients with tracheobronchomalacia (TBM). METHODS: Fourteen tracheal specimens obtained from 2005 to 2018 were used in this study. Surgical resection specimens from patients with TBM and tracheal stenosis (TS) were compared with control tracheal specimens obtained from autopsy cases. We investigated the messenger RNA expression of genes encoding fibroblast growth factor (FGF) binding protein 2 (FGFBP2), FGF receptor R3 (FGFR3), interleukin-1ß (IL1ß), tumor growth factor-ß1 (TGFß1), tissue inhibitor of metalloproteinases 1 (TIMP1), and intercellular adhesion molecule 1 (ICAM1) as well as established markers of airway inflammation including interferon-γ (IFNγ) and tumor necrosis factor (TNF). The relative expression of target transcripts was assessed by quantitative real-time polymerase chain reaction. A histologic examination of the same resected airway specimens was performed on formalin-fixed paraffin-embedded tissue sections. RESULTS: FGFBP2 and FGFR3 showed higher expression in TBM compared with TS and control groups (P < .05 and P < .01, respectively). Furthermore, both TGFß1 and TIMP1 were elevated in TBM patients compared with control subjects (P < .05). Conversely ICAM1 was downregulated in TBM versus TS and control subjects (P < .05). IL1ß, IFNγ, and TNF were increased in TBM, although it did not achieve statistical significance. Histologically compared with control airways both TBM and TS demonstrated submucosal fibrotic changes, with TBM additionally demonstrating alterations in elastin fiber quality and density in the posterior membrane. CONCLUSIONS: Significant changes in gene expression are observed in the tracheal walls of patients with TBM and TS compared with control subjects.

Quantification of Inflammatory Markers in Laryngotracheal Stenosis.

Objectives (1) Develop a novel method for serial assessment of gene and protein expression in laryngotracheal stenosis (LTS). (2) Assess cytokine expression and determine an immunophenotype in LTS. Study Design A matched comparison of endolaryngeal brush biopsy samples from laryngotracheal scar and normal airway. Setting Tertiary care hospital, 2015-2016. Methods Brush biopsy specimens of laryngotracheal scar and normal trachea were obtained from 17 patients with LTS at the time of operating room dilation and were used for protein and RNA extraction. Gene expression of the TH1 cytokine interferon γ (INF-γ), TH2 cytokine interleukin 4 (IL-4), transforming growth factor ß, and collagen 1 (Coll1) was quantified with quantitative real-time polymerase chain reaction. Cytokine analysis was performed with flow cytometry with a cytometric bead array. Results LTS specimens demonstrated a 13.68-fold increase in Coll1 gene expression versus normal ( P < .001, N = 17). Additionally, IL-4 gene expression showed a 3.76-fold increase ( P < .001, N = 17) in LTS scar. When stratified into iatrogenic LTS and idiopathic subglottic stenosis cohorts, INF-γ gene expression was significantly increased in idiopathic subglottic stenosis ( P = .011). Soluble cytokine measurements were below the limit of detection for reliable quantification and thus could not be assessed. Conclusions Brush biopsies from LTS samples can be successfully utilized for RNA extraction and demonstrate the expected increase in Coll1 gene expression associated with LTS. Preliminary gene expression suggests that abnormal collagen production may be mediated by the TH2 cytokine IL-4 and that increased INF-γ expression may represent a key difference between iatrogenic LTS and idiopathic subglottic stenosis. Further analysis of soluble cytokines is needed to confirm these findings.

Congenital tracheobronchial stenosis.

Congenital tracheobronchial stenosis is a rare disease characterized by complete tracheal rings that can affect variable lengths of the tracheobronchial tree. It causes high levels of morbidity and mortality both due to the stenosis itself and to the high incidence of other associated congenital malformations. Successful management of this complex condition requires a highly individualized approach delivered by an experienced multidisciplinary team, which is best delivered within centralized units with the necessary diverse expertise. In such settings, surgical correction by slide tracheoplasty has become increasingly successful over the past 2 decades such that long-term survival now exceeds 88%, with normalization of quality of life scores for patients with non-syndrome-associated congenital tracheal stenosis. Careful assessment and planning of treatment strategies is of paramount importance for both successful management and the provision of patients and carers with accurate and realistic treatment counseling.

Frontometaphyseal dysplasia and keloid formation without FLNA mutations.

Frontometaphyseal dysplasia (FMD) is a distinctive sclerosing skeletal dysplasia associated with a number of non-skeletal manifestations including hearing loss, cardiac malformations, and stenosis, particularly of the upper airway and urinary tract. Some, but not all, patients have mutations in FLNA causing the condition. Consonant with the X chromosomal location of FLNA males are generally more severely affected than females. FLNA mutations can be detected in 82% of affected males. We describe seven patients (one male, six females) all of whom have the major clinical and radiological features of FMD, but without detectable mutations in FLNA. The females in our cohort are affected to a similar degree as is usually found in males. In addition, all patients have marked keloid formation at various body sites, including the eye, from an early age. Other features that may indicate a different etiology in these patients are the increased frequency of cleft palate, Robin sequence, tracheal stenosis, and mild intellectual disability, which all occur in three of more patients in the present group. All patients are isolated. We hypothesize that the presently reported patients represent further evidence that phenotypes strongly resembling FMD exist that are not accounted for by mutations in FLNA. Since the frequency of several of the manifestations, their sporadic presentations, and the presence of keloid formation differ from the X-linked form of this condition we propose de novo autosomal dominant acting mutations in a gene functionally related to FLNA, underpin this disorder.

Idiopathic subglottic stenosis: a familial predisposition.

Idiopathic subglottic stenosis is a narrowing of the trachea at the level of the cricoid cartilage of unknown etiology. It is a rare condition for which the real incidence has never been established owing to the difficulty of making the diagnosis. Although there is a female preponderance, no familial cases have been reported in the literature. We describe two pairs of sisters as well as a mother and daughter presenting with idiopathic subglottic stenosis. All known causes of tracheal stenosis were excluded, including prolonged intubation, surgery, autoimmune and inflammatory disorders, infection and gastroesophageal reflux disease. These are the first cases reported in the literature that suggest a genetic predisposition for idiopathic subglottic stenosis.

The -509 C/T genotype of TGFbeta1 might contribute to the pathogenesis of benign airway stenosis.

Benign airway stenosis (BAS) is one of the most severe complications of endotracheal intubation. The aim of this pilot study was to compare the frequencies of four polymorphisms of the transforming growth factor (TGF) beta1 gene in patients with BAS due to endotracheal intubation (n = 36) and a control group of intensive care patients who had also undergone endotracheal intubation but did not present BAS (n = 30). One of the studied polymorphisms, the -509 C/T, demonstrated a differential genotype distribution between the affected and the control population: the ratio of heterozygous mutants was significantly (P = 0.0116) higher among the control patients. These data suggest a protective function of the frequent heterozygous C/T genotype against BAS; alternatively, the C/C genotype might be a susceptibility factor for BAS (OR 4.5; 95% CI 1.5123-13.3902). Our findings suggest that, besides other iatrogenic factors, a genetic predisposition might contribute to the pathogenesis of BAS.

Matrix metalloproteinase-1 polymorphism in Taiwanese patients with endobronchial tuberculosis.

Endobronchial tuberculosis (TB) often leads to some degree of tracheobronchial stenosis. Because matrix metalloproteinases (MMPs) play an essential role in tissue remodeling in the airways, we investigated the role of MMP-1 polymorphism in patients with endobronchial TB. One hundred and one cases of pulmonary TB in Taiwanese patients were genotyped for the 1G/2G polymorphism of MMP-1 promoter (-1607 bp). Bronchoscopic examination was performed to determine the presence of endobronchial involvement. Levels of MMP-1 in peripheral blood monocytes and in bronchial biopsies were also determined. 1G genotypes of MMP-1 polymorphism, containing at least one 1G allele, were associated with the presence of endobronchial TB. Using multivariate analysis, 1G genotypes and female gender were independent predictors of the development of endobronchial TB. Endobronchial TB patients with 1G genotypes had a 9.86-fold greater risk of developing tracheobronchial stenosis. IL-1beta increased levels of MMP-1 in peripheral blood monocytes of TB patients with 1G genotypes. MMP-1 activity was also present in the endobronchial TB granuloma from patients with 1G/1G genotype. 1G genotypes of MMP-1 polymorphism were associated with a greater risk of developing tracheobronchial stenosis through up-regulation of MMP-1 activity.

Expression of apoptosis-related genes after fetal tracheal occlusion in rabbits.

BACKGROUND/PURPOSE: Late-gestation lung remodeling is associated with alveolar type II cell apoptosis early in the saccular stage (day 28 in fetal rabbits). Intrauterine tracheal occlusion (TO), a potent stimulus of fetal lung growth and maturation, significantly increases type II cell apoptosis. The aim of this study was to determine the effect of fetal TO on the spatiotemporal expression of key apoptosis-related signaling molecules. METHODS: Tracheal occlusion of fetal rabbits was performed at gestational day 25 (term, 31 days), and apoptotic gene expression was studied between days 26 and 28. RESULTS: At days 26 and 27, the protein levels of Fas and Fas-ligand (FasL) in lung lysates were similar in TO fetuses and sham-operated controls. At day 28, however, synchronous with the onset of TO-induced pulmonary distension and type II cell apoptosis, the FasL protein content was 8-fold higher in TO lungs compared with controls (P < .01), whereas Fas levels were comparable. In contrast, Bax and Bcl-2 protein levels were similar in TO and control fetuses at all time-points. TO significantly increased the cellular concentration of immunoreactive FasL in type II cells and bronchial epithelial Clara cells. Furthermore, bronchoalveolar lavage fluid (BAL) from TO fetuses at day 28 induced significantly more type II cell apoptosis in vitro compared with control BAL, an effect that was inhibited by neutralizing anti-FasL antibody. CONCLUSIONS: Our findings show that TO results in time-specific increase of both cellular and soluble FasL in fetal lungs and implicate the Fas/FasL pathway as a pivotal autocrine and/or paracrine regulator of TO- induced type II cell apoptosis.

Mosaic r(13) resulting in large deletion of chromosome 13q in a newborn female with multiple congenital anomalies.

A newborn female presented with multiple congenital anomalies including facial dysmorphism, agenesis of the corpus callosum, type I laryngeal cleft, tracheal stenosis, bilaterally small kidneys, segmental vertebral anomalies, extranumerary rib, bilateral hip dislocation, digital anomalies, and growth retardation. Newborn aneuploidy detection (NAD) based on interphase fluorescence in situ hybridization (FISH) indicated monosomy 13 in 47 of 200 (23.5%) peripheral blood cells (normal cutoff 8.5% at 95% CI). The follow-up banded metaphase-based analysis of 20 cells revealed a karyotype of 46,XX. The analysis of 30 additional cells revealed one cell to have monosomy 13 and a small ring chromosome. In the abnormal cell line, the ring was positive for whole chromosome paint (wcp) 13 and negative for Rb1 (13q14.3). The ring was detected in 4% of 80 additional metaphases studied by FISH. Therefore, the ring was present in 4% (5/130) of metaphases from peripheral blood. Analysis of buccal cells by FISH indicated the ring was present in 36% of cells. A higher degree of mosaicism (60%) was detected in fibroblast cultures from a skin biopsy. The low-level mosaicism of ring 13 in metaphase cells from peripheral blood would have been missed if the standard 20 GTL-banded metaphases had been analyzed. In this case, a preliminary interphase FISH study had indicated monosomy 13 resulting from a large 13q deletion that included the Rb1 locus. This finding initiated the analysis of additional metaphases by GTL-banding and the analysis of metaphases and interphases by FISH. The clinical presentation of our patient was consistent with reported cases of 13q deletions. In addition, our patient had airway anomalies, including a type I laryngeal cleft and tracheal stenosis, which are previously unreported.

Aortic coarctation, vascular ring, and right aortic arch with aberrant subclavian artery.

Right aortic arch, in all situations, is relatively rare. In association with coarctation and vascular compression, it is extremely rare. We present a patient with a right aortic arch and an aberrant left subclavian artery, in addition to coarctation. This was dealt with through a left thoracotomy by dividing the ligamentum arteriosum and placing a Dacron graft from the ascending aorta to the descending aorta.

Familial cryptic translocation with del 4q34-->qter and dup 12pter-->p13 in sibs with tracheal stenosis: clinical, classical and molecular cytogenetic studies and CGH analyses from archival placental tissues evidencing tertiary trisomy 4 in one abortion specimen.

We report on two retarded half-sibs of different sex and seemingly normal karyotype who had the same syndrome of minor anomalies, heart defect and a distal tracheal stenosis, and who shared a healthy mother. These findings raised suspicions of a cryptic chromosome translocation. A translocation t(4;12)(q34;p13), balanced in the mother and unbalanced in the sibs with loss of terminal 4q and gain of terminal 12p regions, was verified by FISH using whole chromosome painting, subtelomeric and YAC probes. Clinical features could be explained by partial monosomy 4q and partial trisomy 12p. Tracheal stenosis was interpreted as a consequence of the same developmental disturbance leading to esophageal atresia and tracheo-esophageal fistula. It was attributed to the 4q deletion in which esophageal atresia as also respiratory difficulties and airway obstructions had been described. Paraffin-embedded placental tissues were available from three of the five abortions of the mother allowing DNA extraction and comparative genome hybridization (CGH). Two of the abortion specimens had the same der(4)t(4;12)(q34;p13) unbalanced translocation as identified in the sibs. In the third abortion specimen, suspicious of triploidy because of partial hydatidiform mole, CGH uncovered a tertiary trisomy 4 resulting from a 3:1 segregation of the translocation chromosomes and their homologs during maternal meiosis I. Differences in CGH results using DNA generated directly or after DOP-PCR were explained by DNA fragmentation in paraffin-embedded tissues and unequal amplification. Am. J. Med. Genet. 94:271-280, 2000.

[Congenital subglottic laryngeal stenosis in 2 brothers with chondrodysplasia syndrome (Keutel-Gabriel syndrome)]. / Kongenitale subglottische Larynxstenose bei zwei Brüdern mit einem Chondrodysplasiesyndrom (Keutel-Gabriel-Syndrom).

BACKGROUND: Keutel-Gabriel syndrome (chondrodysplasia) is a rare autosomal recessive disease. The patients have characteristic malformations such as midfacial hypoplasia, brachytelephalangia, and hearing loss as leading symptoms. PATIENTS: We report about two brothers with clinical and radiological features of Keutel-Gabriel syndrome. Congenital subglottic laryngeal stenosis was also present in both. In the younger brother an emergency tracheotomy had to be performed. In a staged procedure the stenosis was successfully treated with laryngotracheoplasty according to Cotton. CONCLUSIONS: This is the first description of a congenital subglottic laryngeal stenosis with Keutel-Gabriel syndrome. To avoid long-term tracheotomy, a tracheoplasty with autologous cartilage should be performed.

Congenital tracheal stenosis in Pfeiffer syndrome.

We report an infant with Pfeiffer syndrome (acrocephalosyndactyly type V) and a solid cartilaginous trachea lacking rings. This airway abnormality has been reported in a child with Crouzon syndrome but has not been described in Pfeiffer syndrome.

Geleophysic dysplasia: a storage disorder affecting the skin, bone, liver, heart, and trachea.

Geleophysic dysplasia is characterized by typical facies ("happy natured"), small hands and feet, short stature, hepatomegaly, and progressive cardiac disease. We describe five patients (two of whom are siblings) with this disorder and document its variable expressivity. The facies were strikingly similar with small nose, anteverted nostrils, broad nasal bridge, and long thin upper lip with flat and long philtrum. Behavior, development, and intelligence were normal. Growth delay was noticed during infancy, and the two patients who completed normal puberty had marked short stature (140 and 150 cm), with relatively lean body habitus. The hands and feet were small, with short, plump tubular bones and broad proximal phalanges, associated with marked limitation in motion of fingers and wrists. The liver was enlarged after the age of 3 years. Two patients had mild mitral and tricuspid valve stenosis and one had severe aortic stenosis. The most severely affected child died at 3 1/2 years of age of airway obstruction as a result of progressive tracheal narrowing. Lysosomal storage vacuoles were found in skin epithelial cells from three patients whose skin was examined, and in the tracheal mucosa, liver, cartilage and macrophages of the child who died. The basic defect of this autosomal recessive lysosomal storage disease remains to be determined.