Modeling of a Novel Patient-Based MYO5B Point Mutation Reveals Insights Into MVID Pathogenesis.

Microvillus inclusion disease (MVID) is a congenital disorder that presents with severe secretory diarrhea, typically within the first few hours of life. Inactivating mutations of myosin VB (MYO5B) cause MVID, which usually requires lifetime total parenteral nutrition or small-bowel transplantation.1Thiagarajah J.R. et al.Gastroenterology. 2018; 154: 2045-2059.e6Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar A recent analysis of 114 MYO5B mutations identified in MVID patients showed that most mutations were compound heterozygous.2Aldrian D. et al.J Clin Med. 2021; 10: 481Crossref PubMed Scopus (14) Google Scholar Although previously developed homozygous MYO5B knockout animal models showed success in recapitulating MVID,3Bowman D.M. et al.Cell Mol Gastroenterol Hepatol. 2022; 14: 553-565Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar there is a lack of research into the phenotypes caused by compound heterozygous mutations of MYO5B. Targeted exome sequencing in an MVID patient and both parents identified 2 variants in the MYO5B gene: c.1821delG (p.S608HfsX14), likely a de novo mutation that leads to early truncation, and c.1555G>A (p.G519R) (Supplementary Figure 1A), which was maternally inherited. The patient had copious diarrhea from birth with non-ion gap acidosis and intermittent feeding intolerance. An esophagogastroduodenoscopy and flexible sigmoidoscopy at 3 weeks of age were grossly normal. Biopsy specimens showed villus blunting and abnormal accumulation of periodic acid–Schiff–positive in the enterocytes (Figure 1A). Transmission electron microscopy showed disorganized and shortened microvilli, abnormal accumulation of subapical vesicles, and multivesicular bodies. Brush-border dipeptidyl peptidase 4 and terminal web α-actinin-4 were obscure in the MVID patient tissues (Figure 1B). Intriguingly, A-kinase anchoring protein 350 expression was found around the inclusion-like structure, suggesting that the A-kinase anchoring protein–mediated scaffolding pathway is involved in microvillus inclusion formations. Multiplexed immunofluorescence staining (MxIF) showed MVID characteristics on a single slide of duodenum biopsy specimens using 15 markers, including previously known and unexplored proteins in MVID tissues (Figure 1C and D). Basolateral nutrient transporters other than Na–K–adenosine triphosphatase have not been characterized in MVID tissues.4Knowles B.C. et al.J Clin Invest. 2014; 124: 2947-2962Crossref PubMed Scopus (78) Google Scholar The present MxIF shows a remarkable decrease in glucose transporter 2–expressing enterocytes in the MVID patient with tamoxifen-injected VilCreERT2;Myo5bG519R/flox (MYO5B[G519R]), implicating defective enterocyte maturation that could contribute to malabsorption.5Kravtsov D.V. et al.Am J Physiol Gastrointest Liver Physiol. 2016; 311: G142-G155Crossref PubMed Scopus (33) Google Scholar,6Engevik A.C. et al.Gastroenterology. 2018; 155: 1883-1897.e10Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar Consistent with reports from MVID patient tissues with other MYO5B mutations,5Kravtsov D.V. et al.Am J Physiol Gastrointest Liver Physiol. 2016; 311: G142-G155Crossref PubMed Scopus (33) Google Scholar,6Engevik A.C. et al.Gastroenterology. 2018; 155: 1883-1897.e10Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar apical nutrient transporters, such as sodium-dependent glucose transporter 1 (SGLT1) and sodium hydrogen antiporter (NHE3), were internalized, and brush-border markers CD10 and Ezrin scarcely were detected in the enterocytes from the patient with the MYO5B(G519R) mutation.4Knowles B.C. et al.J Clin Invest. 2014; 124: 2947-2962Crossref PubMed Scopus (78) Google Scholar The colocalization of MYO5B with RAB11A in internal puncta in the MYO5B(G519R) patients was different from previous MVID patients with MYO5B(P660L)4Knowles B.C. et al.J Clin Invest. 2014; 124: 2947-2962Crossref PubMed Scopus (78) Google Scholar or MYO5B-deficient mouse models,7Schneeberger K. et al.Proc Natl Acad Sci U S A. 2015; 112: 12408-12413Crossref PubMed Scopus (58) Google Scholar,8Weis V.G. et al.Cell Mol Gastroenterol Hepatol. 2016; 2: 131-157Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar in which MYO5B was separated from RAB11A, which is diffuse in the cytoplasm. The present MxIF panel is useful for documenting deficits in brush-border transporters in congenital diarrheal diseases using single sections of limited biopsy specimens from infants, and this technique will facilitate an accurate diagnosis and provide novel insights into molecular mechanisms of diseases. To understand the molecular basis of MVID pathology resulting from compound heterozygous mutations in MYO5B in this patient, we developed a mouse model mimicking the patient’s genotype. The 1-step 2-cell embryo microinjection technique9Wu Y. et al.Nat Commun. 2019; 10: 2883Crossref PubMed Scopus (16) Google Scholar was used to avoid early lethality in homozygous Myo5bG519R/G519R mice (Figure 2A and Supplementary Figure 1B). Next, we cross-bred the Myo5bG519R/+ mouse with the Villin-CreERT2;Myo5bflox/flox mouse to generate the tamoxifen-inducible, patient-mimicking VilCreERT2;Myo5bG519R/flox mice. In this study, the tamoxifen-injected VilCreERT2;Myo5bG519R/flox mice are referred to as Myo5b(G519R). After a single tamoxifen injection, Myo5b(G519R) mice showed a 19% body weight decrease by day 4, similar to that in VilCreERT2;Myo5bflox/flox (Myo5bΔIEC) mice (Figure 2B). The Myo5b(G519R) mouse ileum and colon indicated a severe watery diarrhea phenotype (Figure 2C). Tamoxifen-treated, Cre-lacking Myo5bG519R/+ mice, which represent the patient’s mother’s genotype, as well as treated VilCreERT2;Myo5bflox/+, displayed healthy intestinal phenotypes (Figure 2B and C) and were used as controls. Myo5b(G519R) intestines showed periodic acid–Schiff–positive vesicles, disordered brush-border structures, microvillus inclusions, and subapical accumulation of MYO5B together with Rab11a, recapitulating the MVID patient’s phenotype (Figure 2D–G). These observations indicate that the Myo5b(G519R) strain phenocopies the MVID patient. Important apical sodium transporters SGLT1, apical sodium-dependent bile acid transporter, and NHE3 were internalized away from the apical membrane in Myo5b(G519R) enterocytes (Supplementary Figure 2A–C), consistent with limited absorption of sodium and water, resulting in watery diarrhea. The actinin-4+ terminal web structure was disrupted (Supplementary Figure 2F), and the expanded SGLT1 localization with lysosome-associated membrane protein 1+ lysosomes in the Myo5b(G519R) enterocytes (Supplementary Figure 2A) suggests degradation of mislocalized proteins, similar to the expanded RAB7+ vesicles in the MVID patient tissues. The localization and function of crypt cystic fibrosis transmembrane conductance regulator were intact in Myo5b(G519R) duodenum (Supplementary Figure 2C and D). Üssing chamber experiments showed that cystic fibrosis transmembrane conductance regulator–dependent Isc, which represents chloride secretion, was up-regulated in steady-state Myo5b(G519R) mouse duodenum. However, bicarbonate secretion, which is likely predominate in villi, showed no significant differences at baseline, after NHE3 inhibition, or forskolin-stimulated conditions (Supplementary Figure 2E). Myo5b(G519R) expanded PCNA+ crypts and sustained subapical OLFM4 signals in upper crypt and lower villus, suggesting an expansion of immature cells (Supplementary Figure 3A and B). EdU+ cells reached 90% of the total epithelial height within 24 hours, suggesting more rapid shedding of villus enterocytes in Myo5b(G519R) mice than in healthy controls (Supplementary Figure 3C and D). Indeed, SEM images showed immature and disorganized microvilli in Myo5b(G519R) mouse enterocytes (Supplementary Figure 3E). These findings are consistent with a deficit in enterocyte maturation in Myo5b(G519R) mice and in the patient with the MYO5B(G519R) mutation. In summary, we showed that point mutation at MYO5B(G519R) in an MVID patient and a genetically engineered mouse model causes severe diarrhea and the disruption of epithelial microvillus structure. This study shows a rapid scheme for establishment of a novel MVID mouse model, Myo5b(G519R), to study the effects of compound heterozygous MYO5B mutations in vivo, and suggests that the combination of 1-step 2-cell embryo microinjection and Cre/loxp systems is a useful tool for modeling patient-specific monogenic congenital disorders. The authors thank Dr Lane Clarke at the University of Missouri for his expertise in bicarbonate secretion measurements. The authors thank the Vanderbilt Digital Histology (VA Shared Equipment grant 1IS1BX003097) and Vanderbilt Cell Imaging Shared Resources (supported by grants CA68485, DK20593, DK58404, DK59637, and EY08126) for excellent imaging techniques. Generation of Myo5bG519R mice by the Vanderbilt Genome Editing Resource (RRID: SCR_018826) was supported by Cancer Center Support grant CA68485, the Vanderbilt Diabetes Research and Training Center grant DK020593, and the Center for Stem Cell Biology. Paraffin sectioning and histologic staining were performed by the Vanderbilt Translational Pathology Shared Resource supported by the National Cancer Institute/National Institutes of Health Cancer Center Support grant P30 CA68485. Download .docx (.07 MB) Help with docx files Supplementary Material Download .pdf (4.1 MB) Help with pdf files Supplementary Figure 1 Download .pdf (12.16 MB) Help with pdf files Supplementary Figure 2 Download .pdf (23.49 MB) Help with pdf files Supplementary Figure 4