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

doi:10.1053/j.gastro.2020.02.034

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

Animal Science

Research output: Contribution to journal › Article › peer-review

28 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 language	English (US)
Pages (from-to)	2236-2249.e9
Journal	Gastroenterology
Volume	158
Issue number	8
DOIs	https://doi.org/10.1053/j.gastro.2020.02.034
State	Published - Jun 2020

Bibliographical note

Publisher Copyright:
© 2020 AGA Institute

Keywords

Malabsorption
Missense Mutation
Motor Protein
Plasma Membrane

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10.1053/j.gastro.2020.02.034

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Engevik, A. C., Coutts, A. W., Kaji, I., Rodriguez, P., Ongaratto, F., Saqui-Salces, M., Medida, R. L., Meyer, A. R., Kolobova, E., Engevik, M. A., Williams, J. A., Shub, M. D., Carlson, D. F., Melkamu, T., & Goldenring, J. R. (2020). Editing Myosin VB Gene to Create Porcine Model of Microvillus Inclusion Disease, With Microvillus-Lined Inclusions and Alterations in Sodium Transporters. Gastroenterology, 158(8), 2236-2249.e9. https://doi.org/10.1053/j.gastro.2020.02.034

Engevik, AC, Coutts, AW, Kaji, I, Rodriguez, P, Ongaratto, F, Saqui-Salces, M, Medida, RL, Meyer, AR, Kolobova, E, Engevik, MA, Williams, JA, Shub, MD, Carlson, DF, Melkamu, T & Goldenring, JR 2020, 'Editing Myosin VB Gene to Create Porcine Model of Microvillus Inclusion Disease, With Microvillus-Lined Inclusions and Alterations in Sodium Transporters', Gastroenterology, vol. 158, no. 8, pp. 2236-2249.e9. https://doi.org/10.1053/j.gastro.2020.02.034

@article{45db4b92057c414d939f46781516b443,

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

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.",

keywords = "Malabsorption, Missense Mutation, Motor Protein, Plasma Membrane",

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

note = "Publisher Copyright: {\textcopyright} 2020 AGA Institute",

year = "2020",

month = jun,

doi = "10.1053/j.gastro.2020.02.034",

language = "English (US)",

volume = "158",

pages = "2236--2249.e9",

journal = "Gastroenterology",

issn = "0016-5085",

publisher = "W.B. Saunders Ltd",

number = "8",

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TY - JOUR

T1 - Editing Myosin VB Gene to Create Porcine Model of Microvillus Inclusion Disease, With Microvillus-Lined Inclusions and Alterations in Sodium Transporters

AU - Engevik, Amy C.

AU - Coutts, Alexander W.

AU - Kaji, Izumi

AU - Rodriguez, Paula

AU - Ongaratto, Felipe

AU - Saqui-Salces, Milena

AU - Medida, Ramya Lekha

AU - Meyer, Anne R.

AU - Kolobova, Elena

AU - Engevik, Melinda A.

AU - Williams, Janice A.

AU - Shub, Mitchell D.

AU - Carlson, Daniel F.

AU - Melkamu, Tamene

AU - Goldenring, James R.

PY - 2020/6

Y1 - 2020/6

N2 - 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.

AB - 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.

KW - Malabsorption

KW - Missense Mutation

KW - Motor Protein

KW - Plasma Membrane

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ER -

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

Abstract

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