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Desmoplakin and periplakin genetically and functionally contribute to eosinophilic esophagitis

Eosinophilic esophagitis (EoE) is a chronic allergic inflammatory disease with a complex underlying genetic etiology. Herein, we conduct whole-exome sequencing of a multigeneration EoE pedigree (discovery set) and 61 additional multiplex families with EoE (replication set). A series of rare, heterozygous, missense variants are identified in the genes encoding the desmosome-associated proteins DSP and PPL in 21% of the multiplex families. Esophageal biopsies from patients with these variants retain dilated intercellular spaces and decrease DSP and PPL expression even during disease remission. These variants affect barrier integrity, cell motility and RhoGTPase activity in esophageal epithelial cells and have increased susceptibility to calpain-14–mediated degradation. An acquired loss of esophageal DSP and PPL is present in non-familial EoE. Taken together, herein, we uncover a pathogenic role for desmosomal dysfunction in EoE, providing a deeper mechanistic understanding of tissue-specific allergic responses.


Sanger sequencing results of variant compared to non-variant DSP and PPL.
For comparison, the corresponding non-variant sequences are shown below each mutated sequence in DSP and PPL identified in 13 of 62 multiplex families with eosinophilic esophagitis (EoE). The arrow indicates where the variants are in each patient.

Schematic of the DSP and PPL gene loci.
The DSP and PPL variants noted above the gene locus schematics are those identified by whole-exome sequencing (WES) in this study in 13 of 62 multiplex families with eosinophilic esophagitis (EoE) (discovery and replication sets).

Supplementary Figure 5
Molecular expression analysis of esophageal biopsies. a, DSG1 mRNA expression from esophageal biopsies from controls and patients with eosinophilic esophagitis (EoE, inactive and active). Data points represent individual patients (Control, n = 48; Inactive EoE, n = 51; Active EoE, n = 147). Statistics: Control vs. Inactive EoE, >0.9999; Control vs. Active EoE, P < 0.0001; Inactive EoE vs. Active EoE, P < 0.0001. b, peak esophageal eosinophil count (left) and DSP (middle) or PPL (right) mRNA expression of patients with active EoE from non-familial EoE and familial EoE with variants (nonfamilial EoE, n = 115; familial EoE with DSP variants, n = 7; familial EoE with PPL variants, n = 8). Statistics: peak esophageal eosinophil count, P = 0.1374; DSP, P < 0.0001; PPL, P = 0.0028. c, Negative log10 FDR P-value of the Spearman correlation between DSP (left) or PPL (right) mRNA expression and a diagnostic subset of genes from the Eosinophilic Esophagitis Diagnostic Panel (EDP). 28 Red indicates a positive correlation, and blue indicates a negative correlation. The top 10 genes of EDP correlation are labeled, and the 8 common genes between DSP and PPL from their respective top 10 genes of EDP correlation are listed (far right). For a-b, data are presented as mean ± SEM. For a-b, n is the number of biologically independent subjects, and two-tailed P values were determined by the Kruskal-Wallis test followed by a Dunn multiple-comparison test (a) or by the Mann-Whitney U test (b). **P < 0.01 and ***P < 0.001. FDR, false discovery rate; NS, not significant; SEM, standard error of the mean.

Supplementary Figure 6
Histopathologic findings in patients with variants in DSP and PPL.  Table S9. d-e, Immunofluorescence staining of esophageal biopsy sections for DSP (cyan) and PPL (magenta) with DAPI-stained nuclei (blue). Data are representative of three experiments performed in duplicate. Scale bar: 100 μM; d, representative images of sections from normal control individuals, patients with non-familial EoE and a patient with EoE and DSP and PPL variants (individual 1 from family 430) during active and inactive (remission) disease state. e, representative images of sections from patients with EoE and DSP (individual 1 from family 1594) or PPL variants (individual 1 from family 1029) during inactive (remission) disease state with counterstain by E-cadherin (red) (less perturbed during the inactive EoE disease state). f-g, quantification of DSP (f) and PPL (g) fluorescence staining intensity. Data are representative of four experiments performed in duplicate. Statistics (versus Control): f, non-familial EoE (Inactive, P = 0.9792; Active, P = 0.0002); familial EoE with variants, (Inactive, P = 0.0006; Active, P < 0.0001); g, nonfamilial EoE (Inactive, P = 0.1392; Active, P < 0.0001); familial EoE with variants, (Inactive, P < 0.0001; Active, P < 0.0001). For b, f and g, data are presented as mean ± SEM. For a-c and f-g, two-tailed P values were determined by the following tests: a and c, Spearman's rank correlation coefficient (Benjamini-Hochberg correction was applied for multiple testing); b, Kruskal-Wallis test followed by a Dunn multiple-comparison test; and f-g, one-way ANOVA test followed by a Dunnett's multiple-comparison test. *P < 0.05, **P < 0.01 and ***P < 0.001. DSP, desmoplakin; PPL, periplakin; FDR, false discovery rate; EI, eosinophilic inflammation; EA, eosinophilic abscess; ESL, eosinophilic surface layering; SEA, surface epithelial alteration; BZH, basal zone hyperplasia; DIS, dilated intercellular spaces; DEC, dyskeratotic epithelial cells; LPF, lamina propria fibrosis; SEM, standard error of the mean. were determined by the two-way ANOVA test followed by a Holm-Sidak's multiple comparisons test (c-d) or one-way ANOVA test followed by a Dunnett's multiple-comparison test (e). *P < 0.05, **P < 0.01 and ***P < 0.001. WT, wild-type; KO, knockout; MW, molecular weight marker; SEM, standard error of the mean. a-e, two-tailed P values were determined by the unpaired t test (a) or one-way ANOVA test followed by a Dunnett's multiple-comparison test (b-e). *P < 0.05, **P < 0.01 and ***P < 0.001. DSP, desmoplakin; PPL, periplakin; SEM, standard error of the mean.    *The Multiz-Alignment counts the number of mammalian species included in the UCSC Genome Browser whose reference allele matches the observed amino acid variant. # Algorithms (PhyloP, SiPhy and GERP++) include information concerning known evolutionary conservation at the level of DNA base pairs.

Supplementary Table 3. Algorithms predicting molecular pathogenicity for all DSP and PPL variants included in this study
Categorical predictions for each algorithm are summarized in Table S5. "C"=Conserved, "NC"=Not Conserved. Abbreviations: NA, not applicable.   Table S5. SIFT: "T"=Tolerated, "D"=Damaging. PolyPhen2