Neonatal-Onset Chronic Diarrhea Caused by Homozygous Nonsense WNT2B Mutations

Abstract

Homozygous nonsense mutations in WNT2B were identified in three individuals from two unrelated families with severe, neonatal-onset osmotic diarrhea after whole-exome sequencing was performed on trios from the two families. Intestinal biopsy samples from affected individuals were used for histology and immunofluorescence and to generate enteroids ex vivo. Histopathologic evaluation demonstrated chronic inflammatory changes in the stomach, duodenum, and colon. Immunofluorescence demonstrated diminished staining for OLFM4, a marker for intestinal stem cells (ISCs). The enteroids generated from WNT2B-deficient intestinal epithelium could not be expanded and did not survive passage. Addition of CHIR-99021 (a GSK3A and GSK3B inhibitor and activator of canonical WNT/β-CATENIN signaling) could not rescue WNT2B-deficient enteroids. Addition of supplemental recombinant murine WNT2B was able to perpetuate small enteroids for multiple passages but failed to expand their number. Enteroids showed a 10-fold increase in the expression of LEF1 mRNA and a 100-fold reduction in TLR4 expression, compared with controls by quantitative RT-PCR, indicating alterations in canonical WNT and microbial pattern-recognition signaling. In summary, individuals with homozygous nonsense mutations in WNT2B demonstrate severe intestinal dysregulation associated with decreased ISC number and function, likely explaining their diarrheal phenotype. WNT2B deficiency should be considered for individuals with neonatal-onset diarrhea.

Keywords: CODE; Lgr5; OLFM4; TLR4; WNT2B; congenital diarrhea and enteropathy; diarrhea; intestinal stem cells.

Figure 1

Individual Growth Charts and Sequencing (A) I-1 growth charts (upper panel, blue lines) and I-2 growth charts (lower, red lines). (B) Sequencing chromatograms for subject S1 along with maternal and paternal sequencing. (C) Pedigrees for I-1 and I-2 (left) and I-3 (right). ? = not known, sequencing not done.

Figure 2

Gastrointestinal Histology Biopsies from endoscopy or colonoscopy were taken from I-1 and I-2. Results are H&E-stained segments from the body of the stomach, proximal duodenum, and right colon. Note loss of crypt architecture and predominance of inflammatory cells in WNT2B-deficient samples.

Figure 3

OLFM4 Localization in WNT2B-Mutated Duodenal Crypts (A) Duodenal sections from I-1 compared with control duodenum. Control samples uniformly demonstrated OLFM4 localization at crypt bases similar to depicted section. Representative WNT2B-deficient samples were selected, with I-1 sample 1 showing no OLFM4 and I-1 sample 2 showing some OLFM4. Blue, DAPI; green, auto-fluorescence; Tx red, OLFM4. 100× mag. (B) Mean fluorescence intensity of crypt bases. Crypt base was identified using the DAPI channel, then selected base of crypt was analyzed for Texas red fluorescence. Error bars represent 25^th and 75^th quartiles. p value for unpaired t test with Walsh adjustment.

Figure 4

Intestinal Enteroids Derived from Biopsy Samples and Grown in Matrigel with Standard Enteroid Media (A–C) Representative images of enteroids from (A) a healthy control individual and from (B) WNT2B-deficient individual (I-2) after 2–3 days and (C) after 4–5 days, when enteroids have collapsed. (D–F) Representative enteroids from WNT2B-deficient individual (I-1) (D) treated with standard media, (E) treated with CHIR-99021, or (F) treated with recombinant murine WNT2B (rmWNT2B). Scale bars are shown at 200 μm.

Figure 5

Comparison of Enteroid Cultures in Standard Enteroid Media (A) Stitched low-magnification comparison of enteroid cultures from healthy control individual (left) versus WNT2B-deficient individual (I-1) treated with recombinant murine WNT2B (rmWNT2B, right) at passage 4. Scale bars are shown at 2 mm. (B) Enteroid size comparison between healthy control samples and WNT2B-deficient enteroids treated with WNT2B. Boxplot is max and min numbers with 75^th and 25^th quartiles. p value for unpaired t test with Walsh adjustment. (C) Semiquantitative analysis showing exponential expansion of enteroid numbers from the control individual scored for the number of wells at each passage compared with enteroids from the WNT2B-deficient individual, which survived each passage but failed to expand in number.

Figure 6

WNT2B Mutant Enteroids Demonstrate Altered Expression of βCATENIN-Target and Inflammatory Genes Duodenal enteroid cultures from I-1 were cultured with recombinant murine WNT2B and gene expression assessed by qRT-PCR. Relative expression is displayed as compared with control enteroids. Expression was normalized to GAPDH.