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 ANTXR2KO 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.

Molecular Effects of Mutations in Human Genetic Diseases

“…The eight papers published in this SI described case studies that may provide a useful framework for understanding the general molecular defects underlying a broad and diverse spectrum of human diseases. These included multifactorial or polygenic disorders, such as lung cancer (OMIM*612052) and osteoporosis (OMIM*166710) [ 1 , 2 ], or monogenic disorders, such as Long QT Syndrome 1 (LQT; OMIM*192500), Hyaline fibromatosis syndrome (OMIM*228600), Dystrophic epidermolysis bullosa (RDEB; OMIM*226600), Xeroderma Pigmentosum C Phenotype (XP-C; OMIM*278720), and MTHFR deficiency (OMIM*236250) [ 3 , 4 , 5 , 6 , 7 ].…”

“…Studying the effects of a genetic variant can have several specific purposes, for instance, the following: (i) evaluating the pathogenicity of a novel variant; (ii) identifying the molecular mechanisms supporting the association of the variants with a disease or with the severity of the disease; (iii) investigating the differential effects of some variants associated with distinct phenotypic expressions of the disease. Two of the SI-published works investigated the role of novel genetic variants in the ANTXR2 and LRP5 genes identified by NGS of affected individuals from selected cohorts [ 2 , 4 ]. Diabasana et al studied the molecular effects of a small nucleotide polymorphism (SNP) of CHRNA5 gene, known to confer susceptibility for lung disease [ 1 ].…”

“…By means of electrode voltage clamp experiments, Oertly et al characterised the electrophysiological properties of the voltage-gated K+ channel KCNQ1 mutant, which was expressed in Xenopus laevis oocytes and compared to oocytes injected with KCNQ1 wild-type [ 3 ]. In the study of Jorik M. van Rijn, the anthrax-toxin assay was performed in patient-derived and control intestinal organoids, to confirm the impact of the p.Ser52Phe missense variant on the physiological ANTXR2 function [ 4 ]. Due to the rarity of the disorder and the difficulties to obtain specimens from patients, the authors used the homology-directed repair-guided gene editing CRISPR/Cas9 approach to create ANTXR2 knock-out (KO) organoids.…”

“…Due to the rarity of the disorder and the difficulties to obtain specimens from patients, the authors used the homology-directed repair-guided gene editing CRISPR/Cas9 approach to create ANTXR2 knock-out (KO) organoids. In this case, the anthrax-toxin assay was applied to select ANTXR2 KO organoids [ 4 ]. Another example of a protein-specific test is provided by Kobaisi et al, who evaluated the expression of XPC, a DNA damage sensor protein, and measured the photosensitivity, and the ability to repair UltraViolet B radiation (UVB)-induced lesions of XP-C cells immortalised from patient primary fibroblasts.…”

“…The significant decrease in multiciliated cells in bronchial epithelia of SNP patients compared to WT patients suggested a SNP-associated alteration of ciliogenesis [ 1 ]. In another SI work, Jorik M. van Rijn et al used second harmonic generation (SHG) imaging, which visualises non-labelled fibrillar proteins such as collagen, and electron microscopy inspection of duodenum and skin sections from ANTXR2-deficient patients [ 4 ]. They demonstrated that loss of ANTXR2-mediated signalling yields abnormal extracellular matrix composition paired with significant collagen VI accumulation in the duodenum [ 4 ].…”