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Investigating the mechanisms by which SMAD6 mutations cause tracheoesophageal birth defects

ABSTRACT: Esophageal atresia (EA) with or without tracheoesophageal fistula (TEF) are tracheoesophageal (TE) birth defects which affect 1 in 3000 live births in the United States. EA occurs when the esophagus fails to form a continuous tube from the oral cavity to the stomach and TEF occurs when there is an abnormal connection between the trachea and esophagus. The etiology of EA/TEF arises when the common foregut tube fails to properly separate into the esophagus and respiratory tract during early embryogenesis. This process is essential for proper organ function and involves complex molecular and morphological processes. As part of a multi-institutional project aiming to identify genes causing tracheoesophageal defects, we identified two EA/TEF patients who presented with different mutations in SMAD6, a BMP signaling inhibitor. We used a CRISPR-based approach to knock down smad6 in frog embryos and observed separation defects of the esophagus and trachea, supporting the conclusion that SMAD6 mutations in humans may cause EA/TEF. To investigate the role of SMAD6 during human development, we used CRISPR to generate human pluripotent stem cell lines with loss-of-function alleles in SMAD6. The human foregut is comprised of an inner layer of endoderm-derived epithelium surrounded by mesenchyme, and it is not known if SMAD6 functions in either or both layers. To elucidate this, we investigated the role of SMAD6 in either foregut epithelium or mesenchyme, or both in combination. Our analysis of human SMAD6-/- tracheoesophageal epithelium and mesenchyme reveals an increase in BMP/TGFβ signaling, consistent with its role as a repressor of these pathways. We also observed patterning defects in both tissue types; SMAD6 mutant endoderm shows increased expression of distal gut tube markers and SMAD6 mutant mesenchyme shows increased expression of cardiac markers. Furthermore, we observed cell differentiation changes, where SMAD6-/- mesenchyme had decreased ability to form CD31-positive vasculature, as well as changes in genes involved in processes such as cell migration and extracellular matrix deposition, which are critical for mesenchyme morphogenesis and differentiation during the process of TE separation. Our results suggest SMAD6 mutations impact the identity and function of human engineered TE tissue. By identifying pathways significant for TE development during early embryogenesis, our results contribute to understanding how patient mutations cause congenital malformations.

ORGANISM(S): Homo sapiens

PROVIDER: GSE305960 | GEO | 2025/12/31

REPOSITORIES: GEO

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Project description:The distal esophagus is an important anatomical area where gastric acid reflux can cause reflux esophagitis (RE), Barrett's esophagus (BE) (intestinal metaplasia), and esophageal adenocarcinoma (EA). The incidence of EA has increased 6-fold in the U.S. since the 1970s, parallel to a significant increase in the prevalence of gastroesophageal reflux diseases (GERD). Although specific host factors might predispose one to disease risk, such a rapid increase in incidence must be predominantly environmental. The cause remains unknown. Our hypothesis is that changes in the foregut microbiome are associated with EA and its precursors, RE and BE in the GERD sequence. We will conduct a case control study to characterize the microbiome in every stage of the GERD sequence as well as analyze the trend in changes in the microbiome along disease progression toward EA. Specific Aim 1. To conduct a comprehensive population survey of the foregut microbiome and demonstrate its association with GERD sequence, by a 16S rRNA gene survey. We will analyze samples of the foregut microbiome at three anatomic loci: mouth, distal esophagus, and gastric corpus. Changes of the microbiota in the distal esophagus will be correlated with the phenotypes. Spatial relationship between the esophageal microbiota and upstream (mouth) and downstream (stomach) foregut microbiotas as well as temporal stability of the microbiome-disease association will also be examined. Specific Aim 2. To define distal esophageal metagenome and demonstrate its association with GERD sequence, by shotgun metagenomic analysis. We will first classify samples of the metagenome into metagenotypes by between-sample k-mer distance and correlate the metagenotypes with the four phenotypes. Subsequent detailed analyses will include pathway-disease and gene-disease associations. DNA viruses and fungi, if identified, also will be correlated with the phenotypes. A significant association between the foregut microbiome composition and GERD sequence, if demonstrated, will be the first step for eventually testing the causal hypothesis that an abnormal microbiome is required for the development of the sequence of phenotypic changes toward EA. If EA and its precursors represent a microbial ecological disease, treating the cause of GERD might become possible, for example, by normalizing the microbiome through use of antibiotics, probiotics, or prebiotics. Causative therapy for GERD could prevent its progression and reverse the current trend of increasing incidence of EA.

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Investigating the mechanisms by which SMAD6 mutations cause tracheoesophageal birth defects

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