Editing Myosin VB Gene to Create Porcine Model of Microvillus Inclusion Disease, With Microvillus-Lined Inclusions and Alterations in Sodium Transporters

Amy C. Engevik, Alexander W. Coutts, Izumi Kaji, Paula Rodriguez, Felipe Ongaratto, Milena Saqui-Salces, Ramya Lekha Medida, Anne R. Meyer, Elena Kolobova, Melinda A. Engevik, Janice A. Williams, Mitchell D. Shub, Daniel F. Carlson, Tamene Melkamu, James R. Goldenring

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)2236-2249.e9
JournalGastroenterology
Volume158
Issue number8
DOIs
StatePublished - Jun 2020

Bibliographical note

Funding Information:
Funding This work was supported by the National Institute of Health (NIH) grants R43 DK109820 to Tamene Melkamu and James R. Goldenring, R01 DK48370, R01 DK70856 and a gift from the Christine Volpe Fund to James R. Goldenring. Amy C. Engevik was supported by NIH F32 DK111101 and KO1 DK12186901. This work was supported by core resources of the Vanderbilt Digestive Disease Center ( P30 DK058404 ), the Vanderbilt-Ingram Cancer Center ( P30 CA68485 ), and imaging supported by both the Vanderbilt Cell Imaging Shared Resource and the Vanderbilt Digital Histology Shared Resource (supported by a VA Shared Equipment Grant 1IS1BX003097 ).

Publisher Copyright:
© 2020 AGA Institute

Keywords

  • Malabsorption
  • Missense Mutation
  • Motor Protein
  • Plasma Membrane

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