Enhanced Collagen Deposition in the Duodenum of Patients with Hyaline Fibromatosis Syndrome and Protein Losing Enteropathy

Abstract

Hyaline fibromatosis syndrome (HFS), resulting from ANTXR2 mutations, is an ultra-rare disease that causes intestinal lymphangiectasia and protein-losing enteropathy (PLE). The mechanisms leading to the gastrointestinal phenotype in these patients are not well defined. We present two patients with congenital diarrhea, severe PLE and unique clinical features resulting from deleterious ANTXR2 mutations. Intestinal organoids were generated from one of the patients, along with CRISPR-Cas9 ANTXR2 knockout, and compared with organoids from two healthy controls. The ANTXR2-deficient organoids displayed normal growth and polarity, compared to controls. Using an anthrax-toxin assay we showed that the c.155C>T mutation causes loss-of-function of ANTXR2 protein. An intrinsic defect of monolayer formation in patient-derived or ANTXR2^KO organoids was not apparent, suggesting normal epithelial function. However, electron microscopy and second harmonic generation imaging showed abnormal collagen deposition in duodenal samples of these patients. Specifically, collagen VI, which is known to bind ANTXR2, was highly expressed in the duodenum of these patients. In conclusion, despite resistance to anthrax-toxin, epithelial cell function, and specifically monolayer formation, is intact in patients with HFS. Nevertheless, loss of ANTXR2-mediated signaling leads to collagen VI accumulation in the duodenum and abnormal extracellular matrix composition, which likely plays a role in development of PLE.

Keywords: ANTXR2; CMG2; extracellular matrix; intestinal lymphangiectasia; organoids; protein losing enteropathy.

Figure 1

Clinical presentation of patients with ANTXR2 deficiency. (A) Hyperpigmentation regions over median malleolus and over the knuckles in both hands of Patient 1. (B) Abdominal X-ray of Patient 1 showing marked bowel distention. (C) H & E stain of duodenal biopsy of Patient 1 showing distortion of architecture and dilation of lymphatics compatible with lymphangicetasia, marked by asterix. (D) Perianal thickened nodules observed in Patient 2. (E) H&E stain of colonic biopsy from Patient 2 showing abnormal architecture and marked hyalinosis. (F) H&E stain of skin biopsy from Patient 2 demonstrating pink homogeneous acellular material throughout the dermis, suggestive of hyaline.

Figure 2

Characteristics of the ANTXR2 mutations. Schematic of (A) the genetic localization of the mutations (indicated in red) and (B) the effect of the mutation in the ANTXR2 protein of Patient 1 (c.155C>T, red bar) and Patient 2 (c.946-1G>A). The splice-site mutation of Patient 2 likely leads to loss of protein (deletion indicated in red). (C) The mutated serine residue of Patient 1 is highly conserved across among the amino acid sequences of various vertebrates, suggesting critical functional relevance.

Figure 3

Normal growth and polarization of ANTXR2-deficient organoids. (A) Microscopic brightfield images of two controls and Patient 1 organoids grown for 7 days in EM. (B) Confocal staining for markers of enterocyte polarization. Actin (yellow) stains the apical membrane, E-cadherin (CDH1, red) stains the basolateral membrane and DAPI (blue) stains the nucleus.

Figure 4

The c.155C>T ANTXR2 mutation results in resistance to anthrax toxin-mediated cell death. (A) A schematic overview of the anthrax toxin mechanism of action, adapted from Deuquet et al. [20]. Anthrax toxin PA83 Is cleaved by furin to PA63, which binds to the vWA domain in ANTXR2. Oligomerization leads to pore formation in the cell membrane, which is promoted by the LF-derivative FP59. This allows internalization of the complex, where it is degraded in the late endosome by low pH. FP59 is then released in the cytosol and causes apoptosis. (B) Hoechst and PI stainings from the anthrax toxin assay performed on control and patient organoids grown on EM. (C) Quantification of the relative PI signal as a measure for cell death. Mean ± SD for n = 2 controls.

Figure 5

Normal monolayer cultures in ANTXR2-deficient organoids. (A) Trans-epithelial electrical resistance measured for control, patient, and ANTXR2^KO organoids. (B,C) Monolayers were grown on EM for 7 days. (B) Microscopic brightfield images. Inserts show examples of blister formation and (C) confocal images for polarization markers, including actin (green, apical membrane), E-cadherin (CDH1, red, basolateral membrane) and DAPI (blue, nucleus).

Figure 6

Abnormal extracellular matrix morphology in duodenum of ANTXR2-deficient patients. (A) Second harmonic generation imaging of duodenal FFPE samples. Red signifies collagen. White arrows point to crypt wall. (B) Duodenal electron microscopy images (×15,000) from control subject (infant evaluated for chronic diarrhea) and Patient 1. Red arrows point to collagen I and yellow arrows point to collagen VI. (C) Collagen VI staining (brown colored) in duodenal sections.