393 Apical Recycling Endosome-Associated Myosin Vb Is Mutated in Microvillus Inclusion Disease and Is Involved in Intestinal Brush Border Development

Abstract

Background & Aims: Microvillus inclusion disease is a rare and fatal congenital enteropathy, presenting with intractable secretory diarrhea shortly after birth. The complete inability to absorb nutrients from intestinal lumen demands total parenteral nutrition, and, eventually, transplantation of the small intestine. MID characteristics varies among patients and generally comprises of villous atrophy and crypt hyperplasia, and, at the cellular level, by the apical brush border atrophy, accumulation of apical proteins, lysosomes and microvilli-like inclusions in the apical cytoplasm of intestinal absorptive cells. Previously we have shown that MID enterocytes display abnormal expression of apical recycling endosomal markers, i.e. Rab11a, FIP-1 (RCP), FIP-5 (Rip11), resulting in a defective apical recycling system in MID. In this study, we aimed to identify the genetic cause and functional consequences that underlie the microvillus inclusion disease. Methods: We screened the genomic DNA of three patients diagnosed with microvillus inclusion disease, their siblings and parents. Biopsies of small intestine from MID and control patients were used to analyze the organization of organelles and localization of proteins involved in intracellular trafficking of brush border proteins. Results: In all MID patients together we have identified two substitutions, one deletion, and two protein truncating mutations in the myosin 5B gene. The MYO5B encodes for an actin filament-binding molecular motor protein that interacts with the small GTPase Rab11a, a marker of recycling endosomes, and thus facilitates the intracellular trafficking of apical proteins towards the apical membrane. We also found aberrant expression and subcellular distribution of myosin Vb protein and other key proteins that interact withmyosin Vb and/or control apical recycling endosome-mediated protein trafficking. Conclusions: The endosomal system that ensures the recycling of brush border proteins, with myosin Vb as a critical regulator, is required to develop and maintain functional apical cell surface in human enterocytes, and perturbations in this can be causally linked to microvillus inclusion disease. Mutations occurring at different positions of MYO5B gene and thus affecting different functional regions of MYO5B protein could explain the diversity of phenotypes present in MID patients. The identification of mutations in MYO5B as the cause for MID brings a major advance in setting the reliable diagnosis, enables the genetic counseling and prenatal screening, as well as paves the way for developing alternative therapeutic strategies.