A Novel mouse model derived from an uncharacterized MVID patient MYO5B mutation

Microvillus inclusion disease (MVID) is a congenital disorder characterized by severe diarrhea that presents within the first days of life. Inactivating mutations of a motor protein, myosin Vb (MYO5B), have been identified as a cause of MVID. Although a majority of MVID patients carry compound heterozygous mutations, all previously established in vivo MVID models carry homozygous mutations or deletion of MYO5B. We identified a novel set of MYO5B mutations in an MVID patient: a G519R point mutation and another that leads to early truncation. To better understand the pathogenesis of MVID from compound heterozygous mutations, we generated a mouse strain carrying this patient-based mutation.To avoid early lethality by a homozygous Myo5b G519R mutation, we employed the one-step two-cell embryo microinjection (OSTCM) method, that ensures at partial WT Myo5b contribution by inducing CRISPR-mediated mutations in only one cell of two-cell stage mouse embryos. A chimeric founder was backcrossed with wildtype, and generated progeny heterozygous for the Myo5b G519R allele. To complete the modeling of the patient’s mutation, we crossbred a sequence-verified N1 Myo5b G519R/+ mouse with the tamoxifen-inducible intestinal epithelial-directed Villin-Cre ERT 2 ;Myo5b flox/ flox mouse to produce VilCre ERT2 ;Myo5b G519R/ flox (Myo5b(G519R)) mice. In the G519R patient’s duodenal biopsy, enterocytes lacked DPP4+ mature brush border and Actinin-4+ terminal web structure. Multiplexed immunofluorescence analyses on the G519R patient biopsy revealed a lack of GLUT2-expressing mature enterocytes and accumulation of MYO5B together with RAB11A in the cytoplasm. Adult Myo5B(G519R) mice demonstrated a severe, watery diarrheal phenotype 4 days after the tamoxifen injection. The Myo5B(G519R) mouse intestine illustrated various characteristic features of MVID, such as microvillus inclusions, villus blunting, and subapical accumulation of PAS+ vesicles in the enterocytes. Like the G519R patient, immunostaining in the small intestine of Myo5b(G519R) mice demonstrated a mis-localization of apical nutrient transporters and cytoplasmic accumulation of Myo5b with Rab11a. Electron microscopy revealed the shortened, disorganized microvilli on enterocytes of both the villus and crypt. The crypts of Myo5b(G519R) mouse small intestine were elongated compared to control, mirrored by an expansion of PCNA+, EdU+, and OLFM4+ epithelial cell regions. This study provides a novel MVID patient-mimicking mouse model with compound heterozygous mutations in MYO5B and demonstrates the utility of combining the OSTCM and Cre/ loxp systems for modeling deadly monogenic congenital diseases, like MVID. NSF GRFP, NIH R01DK128190, RC2DK118640 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.