Dysregulation of the NRG1/ERBB pathway causes a developmental disorder with gastrointestinal dysmotility in humans

Abstract

Hirschsprung disease (HSCR) is the most frequent developmental anomaly of the enteric nervous system, with an incidence of 1 in 5000 live births. Chronic intestinal pseudo-obstruction (CIPO) is less frequent and classified as neurogenic or myogenic. Isolated HSCR has an oligogenic inheritance with RET as the major disease-causing gene, while CIPO is genetically heterogeneous, caused by mutations in smooth muscle-specific genes. Here, we describe a series of patients with developmental disorders including gastrointestinal dysmotility, and investigate the underlying molecular bases. Trio-exome sequencing led to the identification of biallelic variants in ERBB3 and ERBB2 in 8 individuals variably associating HSCR, CIPO, peripheral neuropathy, and arthrogryposis. Thorough gut histology revealed aganglionosis, hypoganglionosis, and intestinal smooth muscle abnormalities. The cell type-specific ErbB3 and ErbB2 function was further analyzed in mouse single-cell RNA sequencing data and in a conditional ErbB3-deficient mouse model, revealing a primary role for ERBB3 in enteric progenitors. The consequences of the identified variants were evaluated using quantitative real-time PCR (RT-qPCR) on patient-derived fibroblasts or immunoblot assays on Neuro-2a cells overexpressing WT or mutant proteins, revealing either decreased expression or altered phosphorylation of the mutant receptors. Our results demonstrate that dysregulation of ERBB3 or ERBB2 leads to a broad spectrum of developmental anomalies, including intestinal dysmotility.

Keywords: Development; Gastroenterology; Genetic diseases; Molecular genetics.

Figure 1. Biallelic ERBB3 and ERBB2 variations in 8 affected individuals from 5 families.

(A) Pedigrees and segregation of ERBB3 (NM_001982.3) and ERBB2 (NM_004448.3) variants. Mutant alleles are indicated in red. (B and C) Schematic representation of ERBB3 (B) and ERBB2 (C) proteins showing the predicted consequences of variants identified in this study. ERBB3 variants previously reported in developmental disorders are indicated below the schematic. The localization of putative phosphorylation sites are indicated by blue arrowheads. Inactive ECD: inactive extracellular domain; pseudo KD: pseudo kinase domain.

Figure 2. Histology of gut specimens from patients with ERBB3 or ERBB2 mutations.

(A and B) Patient F1:II-3. (A) H&E-stained sections showed aganglionosis and hypertrophic nerve fibers (arrow) in the rectum, atrophy/disorganization of the external muscularis in the colon, and ectopic location of myenteric plexuses (arrows) within the external muscularis of the small intestine. (B) Immunohistochemistry for S100 indicated presence of enteric glia and ectopic localization of enteric plexuses (arrows). Lack of SMA staining in the internal muscularis (star) of the small intestine, and normal desmin staining were observed. cKIT staining appeared normal (arrows point to ICCs). (C) Patient F2:II-2 and (D) Patient F2:II-3. H&E-stained sections revealed a short distal agangliononic segment without overt hyperplastic nerves (photos on the left-hand side), followed by a long hypoganglionic segment containing rare scattered ganglion cells (arrows in photos on the right-hand side). (E) Patient F5:II-2. H&E-stained sections showed a lack of myenteric and submucosal ganglion cells and a hyperplasia of nerve fibers in the colon (arrows). Scale bars: 100 μm.

Figure 3. Histology of gut specimens from fetuses with ERBB3 mutations.

(A) Fetus F3:II-1 and (B) fetus F4:II-1, along with age-matched controls (C-16WG and C-18WG, respectively). H&E-stained sections showed total colonic aganglionosis, confirmed by absence of PHOX2B and S100 staining in both fetuses. In the controls, enteric neurons and glia were observed (arrows). SMA staining was normal (arrows). Scale bars: 100 μm.

Figure 4. Histology of skeletal muscles, the anterior horn of the spinal cord and dorsal root ganglia of fetuses with ERBB3 mutations.

(A–C) H&E-stained sections of muscle specimens from F3:II-1 (A, quadriceps femoris muscle at 16 WG), F3:II-3 (B, deltoid muscle at 15 WG), and F4:II-1 (C, psoas muscle at 18 WG). Note the presence of the unequal caliber of muscle fibers with the presence of numerous myotubes characterized by central nuclei (blue arrows) compared with the control muscles (C-16WG, C-15WG, and C-18WG psoas muscles), where muscle fibers are homogeneous in size and density with most nuclei positioned at the periphery of mature fibers. (D) Analysis at the ultrastructural level by electron microscopy shows isolated primary myotubes (M1) in fetus F4:II-1, while in the control, secondary myotubes (M2) were observed apposed to the primary myotube (M1). (E) Immunohistochemical analyses using NCL-MHCs and NCL-MHCf antibodies (staining slow and fast myosin heavy chains, respectively) showed the presence of type I and II myotubes, respectively. (F) Preserved cyto-architecture of cervical spinal cord of fetus F4:II-1. AH: anterior horn; DRG: dorsal root ganglion. (G) Higher magnification of boxed regions in F. At the cellular level, both motoneurons in the anterior horn (blue arrows in AH) and sensory neurons in the dorsal root ganglia (blue arrows in DRG) displayed normal morphology and density. Scale bars: 100 μm in A–C, E–G; 2 μm in D.

Figure 5. Investigation of the cell type specificity of ERBB3/ERBB2 in ENS and ISM development.

(A) Expression of ErbB3 and ErbB2 in different populations identified by scRNA-seq at E15.5 and E18.5. Uniform Manifold Approximation and Projection (UMAP) representations indicate clusters corresponding to progenitors, Schwann cells precursors (SCP), enteric glia, and neurons. Feature plots show expression of ErbB3 and ErbB2. Color bars indicate expression level with maximum cut off at the 90th percentile. (B) H&E staining and immunohistochemistry for SMA (green) and TUJ1 (red) on small intestine and colon sections from WT and Wnt1::Cre Erbb3^lox/lox (Mutant) embryos at E17.5 show normal ISM layer organization. In each case, 10 to 20 sections of n = 3 controls and n = 3 mutants were analyzed. Scale bars: 20 μm.

Figure 6. Decreased ERBB3 expression due to the c.3297delG variant.

(A) ERBB3 mRNA expression level in fibroblasts determined by RT-qPCR. F2:II-2 and F2:I-2 represent the patient and his mother, respectively. The relative abundance of the ERBB3 amplicon was normalized to the GUSB internal control and presented relative to that of cells from the control. Data represent mean ± SEM (in black) of relative gene expression of n = 6 independent biological replicate experiments (in gray) performed in triplicate. Statistical differences were determined using a 2-tailed t test on ΔCt values and Holm-Bonferroni correction. NS: P > 0.05. **P < 0.01, ***P < 0.001. (B) Sanger sequencing identified the c.3297delG variant in genomic DNA (gDNA) extracted from peripheral blood samples and in cDNA amplified from the patient and his mother’s fibroblast, and its absence in a control.

Figure 7. Conservation and 3D analysis of ERBB3 and ERBB2 variants.

(A and B) Conservation of the relevant amino acids in human (h), mouse (m), chick (c), and zebrafish (z); asterisks indicate conserved residues in ERBB3 (A) and ERBB2 (B). (C) 3D modeling showing the positions of the identified variants on the crystal structure of the pseudo-kinase domain of ERBB3 (left side) and structural analysis of WT and mutants (right side). Hydrogen bonds are shown in green. Mutations are indicated in different colors (red: p.(Thr787Pro); blue: p.(Val899Met); yellow: p.(Thr873Ser); pink: p.(Gln932Arg)) and presented by family. The WT proteins are depicted in the left column and the mutant versions on the right. Note the p.(Thr787Pro) variant is expected to cause a loss of 2 hydrogen bonds between the Thr787 and the Cys740 residues, while p.(Thr873Ser) and p.(Gln932Arg) are predicted to add a hydrogen bond between the substituted amino acids and the Gln865 or the Pro933 residues, respectively. (D) 3D modeling showing the position of the identified variant on the crystal structure of the kinase domain of ERBB2 (left side) and structural analysis of WT and mutant (gray: p.(Ala710Val); right side). Note absence of modifications.

Figure 8. Functional consequences of ERBB3 and ERBB2 missense variants.

(A) Subcellular localization of ERBB3 and ERBB2 mutant proteins. DAPI (blue), an antibody directed against the tagged ERBB3 (anti-HA, green) or tagged ERBB2 (anti-FLAG, green) receptors and phalloidin (red) were used to visualize subcellular localization of mutant proteins. In each case, 100 cells in each of n = 3 independent experiments were observed. Merge representative images are presented, with fluorescence intensity graphs on the right depicting the colocalization, with red and green lines indicating the corresponding fluorescence of phalloidin and tagged protein, respectively. Variants are presented per family and compared with controls (WT ERBB3 or WT ERBB2). Scale bars: 10 μm. (B–G) Phosphorylation of ERBB3 and ERBB2 analyzed by Western blot, following transfection of Neuro-2a cells with WT or mutant ERBB3- and ERBB2-encoding plasmids. Representative gels (B and E) along with quantifications (C, D, F, and G, respectively) are shown. Actin beta (ACTB) was used as loading control. Cells were untreated (–) or treated (+) with NRG1. ERBB3 mutants are shown in B–D and the ERBB2 mutant in E–G compared with WT proteins (WT). In C, D, F, and G, data represent mean ± SEM. Statistical differences were determined from data of at least n = 3 independent biological replicate experiments using Mann-Whitney tests. After Holm-Bonferroni correction, NS: P > 0.05, *P < 0.05, **P < 0.01. MW: molecular weight.