Amy C Engevik1, Alexander W Coutts2, Izumi Kaji3, Paula Rodriguez2, Felipe Ongaratto2, Milena Saqui-Salces4, Ramya Lekha Medida4, Anne R Meyer3, Elena Kolobova3, Melinda A Engevik5, Janice A Williams3, Mitchell D Shub6, Daniel F Carlson2, Tamene Melkamu2, James R Goldenring7. 1. Department of Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee; The Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, Tennessee. Electronic address: amy.c.engevik@vumc.org. 2. Recombinetics Inc, Saint Paul, Minnesota. 3. Department of Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee; The Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, Tennessee. 4. Department of Animal Science, University of Minnesota, Saint Paul, Minnesota. 5. Baylor College of Medicine and Texas Children's Hospital, Houston, Texas. 6. Phoenix Children's Hospital and University of Arizona College of Medicine-Phoenix, Phoenix, Arizona. 7. Department of Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee; The Epithelial Biology Center, Vanderbilt University School of Medicine, Nashville, Tennessee; Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee; Nashville Veterans Affairs Medical Center, Nashville, Tennessee.
Abstract
BACKGROUND & AIMS: Microvillus inclusion disease (MVID) is caused by inactivating mutations in the myosin VB gene (MYO5B). MVID is a complex disorder characterized by chronic, watery, life-threatening diarrhea that usually begins in the first hours to days of life. We developed a large animal model of MVID to better understand its pathophysiology. METHODS: Pigs were cloned by transfer of chromatin from swine primary fetal fibroblasts, which were edited with TALENs and single-strand oligonucleotide to introduce a P663-L663 substitution in the endogenous swine MYO5B (corresponding to the P660L mutation in human MYO5B, associated with MVID) to fertilized oocytes. We analyzed duodenal tissues from patients with MVID (with the MYO5B P660L mutation) and without (controls), and from pigs using immunohistochemistry. Enteroids were generated from pigs with MYO5B(P663L) and without the substitution (control pigs). RESULTS: Duodenal tissues from patients with MVID lacked MYO5B at the base of the apical membrane of intestinal cells; instead MYO5B was intracellular. Intestinal tissues and derived enteroids from MYO5B(P663L) piglets had reduced apical levels and diffuse subapical levels of sodium hydrogen exchanger 3 and SGLT1, which regulate transport of sodium, glucose, and water, compared with tissues from control piglets. However, intestinal tissues and derived enteroids from MYO5B(P663L) piglets maintained CFTR on apical membranes, like tissues from control pigs. Liver tissues from MYO5B(P663L) piglets had alterations in bile salt export pump, a transporter that facilitates bile flow, which is normally expressed in the bile canaliculi in the liver. CONCLUSIONS: We developed a large animal model of MVID that has many features of the human disease. Studies of this model could provide information about the functions of MYO5B and MVID pathogenesis, and might lead to new treatments.
BACKGROUND & AIMS:Microvillus inclusion disease (MVID) is caused by inactivating mutations in the myosin VB gene (MYO5B). MVID is a complex disorder characterized by chronic, watery, life-threatening diarrhea that usually begins in the first hours to days of life. We developed a large animal model of MVID to better understand its pathophysiology. METHODS:Pigs were cloned by transfer of chromatin from swine primary fetal fibroblasts, which were edited with TALENs and single-strand oligonucleotide to introduce a P663-L663 substitution in the endogenous swineMYO5B (corresponding to the P660L mutation in humanMYO5B, associated with MVID) to fertilized oocytes. We analyzed duodenal tissues from patients with MVID (with the MYO5BP660L mutation) and without (controls), and from pigs using immunohistochemistry. Enteroids were generated from pigs with MYO5B(P663L) and without the substitution (control pigs). RESULTS: Duodenal tissues from patients with MVID lacked MYO5B at the base of the apical membrane of intestinal cells; instead MYO5B was intracellular. Intestinal tissues and derived enteroids from MYO5B(P663L) piglets had reduced apical levels and diffuse subapical levels of sodium hydrogen exchanger 3 and SGLT1, which regulate transport of sodium, glucose, and water, compared with tissues from control piglets. However, intestinal tissues and derived enteroids from MYO5B(P663L) piglets maintained CFTR on apical membranes, like tissues from control pigs. Liver tissues from MYO5B(P663L) piglets had alterations in bile salt export pump, a transporter that facilitates bile flow, which is normally expressed in the bile canaliculi in the liver. CONCLUSIONS: We developed a large animal model of MVID that has many features of the human disease. Studies of this model could provide information about the functions of MYO5B and MVID pathogenesis, and might lead to new treatments.
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