Overall, 22 controls, mean age 32.6 (25–48), 72.7% female, and 37 achalasia patients, mean age 50.3 (25–82), 59.4% female, were included. The controls all had normal endoscopy and 20 had normal motility on HRM. The achalasia cohort entailed 21 with type I achalasia (8/21, 38.0% with dilated esophagus) and 14 with type II achalasia (6/14, 42.9% with dilated esophagus). Table 1 describes subject characteristics by analysis.
Transcriptomic analyses reveal changes in gene expression levels in the proximal and distal esophagus of achalasia patients
We performed RNA-seq on esophageal mucosal biopsies from the proximal and distal esophagus of 14 achalasia and 8 healthy controls to identify gene targets that are dysregulated in achalasia (Table 1). Biopsies from the proximal and distal esophagus of these subjects were processed, sequenced, and analyzed separately to determine if regional differences are observed within subjects. Examples of the histology of the biopsies collected and processed are shown in Fig. 1. Differential gene expression was determined compared to healthy controls. Overall, 706 genes were significantly changed in the distal esophagus and 1896 genes were significantly changed in the proximal esophagus (false discovery rate-adjusted p value < 0.05). The gene expression profiles of each sample were visualized and compared using heat maps (Figs. 2a, 3a) and volcano plots (Figs. 2b, 3b). PCA was used to evaluate similarities and differences between controls and achalasia patients, and to determine if each set of subjects can be grouped (Fig. 2c, 3c). As shown in Fig. 2c, PCA plot shows apparent clustering of the achalasia subjects in the distal esophagus, separated from the controls. Data obtained from the proximal esophagus shows some clustering of achalasia patients and controls, despite more variability within each group (Fig. 3c).
Transcriptomic analysis identifies pathways differentially regulated in achalasia subjects versus healthy controls
After filtering differentially expressed genes (DEGs) between controls and achalasia patients using a threshold of fold change in expression of > 0.9 or < 0.9, we identified 65 DEGs in distal esophagus (31 up-regulated and 34 down-regulated) (Fig. 2d) and 120 DEGs in proximal esophagus (81 up-regulated and 39 down-regulated) (Fig. 3d). Gene Set Enrichment Analysis using Metascape Figs. 2e,f, Figs. 3e,f) and GSEA (Supplementary Figs. 1, 2) were performed using the compilation of genes differentially expressed from achalasia compared to controls. The distal esophageal mucosa of achalasia subjects showed increased gene expression in 7 pathways including those related to cellular response to cytokine stimulus and defense response to virus, when compared to the controls (Fig. 2e). Decreased gene regulation in 6 pathways including those linked to skeletal muscle organ development, G alpha (i) signaling events and regulation of ERK1 and ERK2 cascade and axon development were observed in the distal esophageal mucosa from achalasia patients versus controls (Fig. 2f). On the other hand, mucosa from the proximal esophagus of achalasia had increased gene regulation in 12 pathways including those associated with leukotriene D4 metabolic process, degradation of extracellular matrix, NGF stimulated transcription, negative regulation of epithelial differentiation, positive regulation of tyrosine phosphorylation of STAT protein, angiogenesis, and Notch signaling pathway (Fig. 3e). Finally, down-regulated gene regulation in 4 pathways including ECM glycoproteins and matrisome associated were detected in proximal esophageal mucosa of achalasia patients (Fig. 3f).
Regional differences in gene expression are observed in the proximal versus distal esophagus of achalasia patients
Our RNA-seq analyses showed differences in the number of DEGs in the distal and proximal esophageal mucosa from achalasia subjects (Figs. 2d and 3d) resulting in differential enrichment of pathways in each region (Figs. 2e,f, 3e,f). As shown in Fig. 4a, only 23 DEGs were found to be common between the distal and proximal esophagus of achalasia patients. 13 of these genes were up-regulated (Fig. 4b) and 10 were down-regulated (Fig. 4c). Examples of common DEGs include CPA3, MAMDC2, and CAPN6. 42 DEGs were exclusive to the distal esophageal mucosa of achalasia patients (Fig. 4a): 18 of these DEGs were up-regulated (Fig. 4b) and 24 were down-regulated (Fig. 4c). The most significantly DEGs only in the distal esophagus include IL-33, IFNε, LOX, and JUN. On the other hand, 97 genes were uniquely differentially expressed in the proximal esophageal mucosa of achalasia patients (Fig. 4a): 68 of these genes were up-regulated (Fig. 4b) and 29 were down-regulated (Fig. 4c). This includes change in expression in CDH16, HES5, IGFBP3 and FGF14.
Quantitative polymerase chain reaction (qPCR) validates DEGs in achalasia when compared to controls
To validate the DEGs identified by RNA-sequencing, we performed quantitative PCR (qPCR) on esophageal mucosal samples in a distinct cohort from that used for RNA-seq (4 controls, 23 achalasia patients). Table 1 shows the clinical characteristic of the enrolled subjects. Among the genes identified by RNA-sequencing, we selected 5 genes to be validated by qPCR: the mast cell specific protease carboxypeptidase (CPA3), the cytokine interleukin 33 (IL-33), the type 1 interferon family member interferon epsilon (IFNε), the antiviral response gene MAM domain containing 2 (MAMDC2) and the cell adhesion molecule cadherin 16 (CHD16). As shown in Fig. 5a, CPA3 mRNA expression levels were significantly up-regulated in the distal mucosa of achalasia subjects compared to controls, but not in the proximal esophagus. On the other hand, the cytokine IL-33, known to activate target cells such as mast cells and type 2 innate lymphoid cells15, had significant increased mRNA expression levels in the proximal esophageal mucosa of achalasia patients, but this change was not significant in the distal esophagus (Fig. 5b). We also observed that achalasia patients had a strong increase in mRNA expression levels of the type I interferon IFNε, in both the distal and proximal esophageal mucosa compared to controls (Fig. 5c). Interestingly, a significant increase in CDH16, an atypical member of the cadherin family16, was seen in both the proximal and distal esophageal mucosa of achalasia subjects compared to controls (Fig. 5d). Finally, we observed a significant decrease in MAMDC2 in the proximal esophageal mucosa of achalasia patients compared to controls, but this change was not significant in the distal esophagus (Fig. 5e).
Infiltration of intraepithelial leukocytes is detected in the esophageal epithelium of achalasia patients
Our RNA-seq and qPCR analyses showed changes in many genes associated with inflammation and immune cell infiltration in achalasia. We first examined the presence of immune cells by performing immunofluorescence to detect the leukocyte marker CD45 in human esophageal mucosal biopsies from achalasia and healthy subjects. As shown in Fig. 6, immunostaining and scoring for CD45 showed increased infiltration of intraepithelial leukocytes in the distal esophageal mucosa of achalasia subjects (Fig. 6b,c), compared to controls (Fig. 6a,c). A significant increase in the recruitment of intraepithelial leukocytes was also observed in the proximal esophageal mucosa of achalasia patients (Fig. 6e,f) compared to controls (Fig. 6d,f).
Distinct changes in gene expression and differentially regulated pathways are observed in the proximal and distal esophagus of type I versus type II achalasia patients
Given that type I achalasia is usually a later phase of disease progression, we next determined differential gene expression between type I or type II achalasia and healthy controls, in both proximal and distal esophagus. As showed in Fig. 7a, we identified 501 DEGs (240 up-regulated and 261 down-regulated) in the distal esophagus of type I achalasia compared to healthy controls. For type II achalasia, we found 144 DEGs in distal esophagus; 77 of these DEGs were up-regulated and 67 DEGs were down-regulated (Fig. 7b). In the proximal esophagus of type 1 achalasia, we identified 329 DEGs (209 up-regulated and 120 down-regulated) (Fig. 8a). On the other hand, the type II achalasia had 294 DEGs (191 up-regulated and 103 down-regulated) in proximal esophagus (Fig. 8b). We then determined the number of DEGs that were common or exclusive to type I and type II achalasia. In distal esophagus, we found 86 DEGs to be common between type I and type II achalasia (Fig. 7c). A total of 415 DEGs were exclusive to type I achalasia (Fig. 7c): 194 DEGs were up-regulated (Fig. 7d) and 221 were down-regulated (Fig. 7e). We found 58 DEGs to be exclusive to type II achalasia (Fig. 7c). Of these 58 genes, 31 were up-regulated (Fig. 7d) and 27 were down-regulated (Fig. 7e). In proximal esophagus, a total of 156 DEGs were common to type I and type II achalasia (Fig. 8c): 110 were up-regulated (Fig. 8d) and 46 were down-regulated (Fig. 8e). A total of 173 DEGs were exclusive to type I achalasia (Fig. 8c). 99 of those genes were up-regulated (Fig. 8b) and 74 were down-regulated (Fig. 8e). Type II achalasia had a total of 138 DEGs (Fig. 8c): 81 genes were up-regulated (Fig. 8d) and 57 genes were down-regulated (Fig. 8e).
Lists of DEGs from type I achalasia and type II achalasia were used to perform Gene Set Enrichment Analysis using Metascape (Figs. 7f,g, 8f,g) and GSEA (Supplementary Figs. 3–6). As shown in Fig. 7f, pathways associated to lymphocyte activation, regulation of cell adhesion, smooth muscle contraction, positive regulation of immune response and focal adhesion were enriched in the distal esophagus of type 1 achalasia. We also found decreased gene regulation of genes associated to pathways related to extracellular matrix, leukocyte tethering or rolling, negative regulation of canonical Wnt signaling and regulation of leukocyte proliferation (Fig. 7g). In the distal esophagus of type II achalasia, we observed increased gene regulation in pathways related to intermediate filament organization and negative regulation of wound healing (Fig. 7f) and decreased expression of genes related to pathways such as fat cell differentiation, NGF stimulated transcription and regulation of system process (Fig. 7g). Analyses performed in proximal esophagus showed the enrichment of pathways related to matrisome, delta Np63 pathway, positive regulation of signaling receptor activity in type I achalasia (Fig. 8f). Decreased gene regulation of genes associated to reactive oxygen species metabolic process, defense response to fungus, matrisome associated and PLC beta mediated events was seen (Fig. 8g). In type II achalasia, increased expression of genes related to peptide cross-linking, organic hydroxy compound transport and transport of small molecules was observed (Fig. 8f). Analyses also showed decreased gene regulation of genes associated to pathways related to negative regulation of secretion, smooth muscle contraction and regulation of angiogenesis (Fig. 8g).