Microvillus Inclusion Disease Research Articles

Alterations in cellular metabolic pathway and epithelial cell maturation induced by MYO5B defects are partially reversible by LPAR5 activation.

Functional loss of the motor protein, Myosin Vb (MYO5B), induces various defects in intestinal epithelial function and causes a congenital diarrheal disorder, microvillus inclusion disease (MVID). Utilizing the MVID model mice, Vil1-CreERT2;Myo5bflox/flox (MYO5B∆IEC) and Vil1-CreERT2;Myo5bflox/G519R (MYO5B(G519R)), we previously reported that functional MYO5B loss disrupts progenitor cell differentiation and enterocyte maturation that result in villus blunting and deadly malabsorption symptoms. In this study, we determined that both absence and a point mutation of MYO5B impair lipid metabolism and alter mitochondrial structure, which may underlie the progenitor cell malfunction observed in MVID intestine. Along with a decrease in fatty acid oxidation, the lipogenesis pathway was enhanced in the MYO5B∆IEC small intestine. Consistent with these observations in vivo, RNA-sequencing of enteroids generated from the two MVID mouse strains showed similar downregulation of energy metabolic enzymes, including mitochondrial oxidative phosphorylation genes. In our previous studies, lysophosphatidic acid (LPA) signaling ameliorates epithelial cell defects in MYO5B∆IEC tissues and enteroids. The present study demonstrated that the highly soluble LPAR5-preferred agonist, Compound-1, improved sodium transporter localization and absorptive function, and tuft cell differentiation in patient-modeled MVID animals that carry independent mutations in MYO5B. Body weight loss in male MYO5B(G519R) mice was ameliorated by Compound-1. These observations suggest that Compound-1 treatment has a trophic effect on intestine with MYO5B functional loss through epithelial cell-autonomous pathways that can accelerate the differentiation of progenitor cells and the maturation of enterocytes. Targeting LPAR5 may represent an effective therapeutic approach for treatment of MVID symptoms induced by different point mutations in MYO5B.

Altered cellular metabolic pathway and epithelial cell maturation induced by MYO5B defects are partially reversible by LPAR5 activation.

This study demonstrates the importance of MYO5B for cellular lipid metabolism and mitochondria in intestinal epithelial cells, a previously unexplored function of MYO5B. Alterations in cellular metabolism may underlie the progenitor cell malfunction observed in microvillus inclusion disease (MVID). To examine the therapeutic potential of progenitor-targeted treatments, the effects of LPAR5-preferred agonist, Compound-1, was investigated utilizing several MVID model mice and enteroids. Our observations suggests that Compound-1 may provide a therapeutic approach for treating MVID.

Intracellular Trafficking Defects in Congenital Intestinal and Hepatic Diseases.

Enterocytes and liver cells fulfill important metabolic and barrier functions and are responsible for crucial vectorial secretive and absorptive processes. To date, genetic diseases affecting metabolic enzymes or transmembrane transporters in the intestine and the liver are better comprehended than mutations affecting intracellular trafficking. In this review, we explore the emerging knowledge on intracellular trafficking defects and their clinical manifestations in both the intestine and the liver. We provide a detailed overview including more investigated diseases such as the canonical, variant and associated forms of microvillus inclusion disease, as well as recently described pathologies, highlighting the complexity and disease relevance of several trafficking pathways. We give examples of how intracellular trafficking hubs, such as the apical recycling endosome system, the trans-Golgi network, lysosomes, or the Golgi-to-endoplasmic reticulum transport are involved in the pathomechanism and lead to disease. Ultimately, understanding these processes could spark novel therapeutic approaches, which would greatly improve the quality of life of the affected patients.

Distinct Treatment Outcomes between MVID Patient-Modeled Mice Suggest Epithelial Cell Maturation Is Influenced by MYO5B Functional State

Background and aims: Microvillus inclusion disease (MVID) is a devastating congenital diarrhea and malnutrition disorder caused by different point mutations in Myosin Vb (MYO5B). Specific genotype-dependent epithelial phenotypes or treatment options have not been fully investigated. Using tamoxifen-inducible epithelial-specific MYO5B-deficient (Myo5bΔIEC) mice, we have reported that MYO5B loss disrupts progenitor cell differentiation and that Lyso-Phosphatidic Acid (LPA) signaling and Notch inhibition can ameliorate the differentiation defects. We recently generated another MVID patient-modeled mouse strain, Vil1-CreERT2;Myo5bflox/G519R (Myo5b(G519R)). This study compared the epithelial pathophysiology and the effects of drug treatment between Myo5b(G519R) and Myo5bΔIEC mice. Methods: The MVID model mice and littermate controls received a single dose of tamoxifen, and their intestinal tissues were harvested on day 4 post-tamoxifen. For treatment: the small chemical LPAR5 agonist, Compound-1, or vehicle were given intraperitoneally once a day. Another set of mice received the Notch2-targeted inhibitor, Nirogacestat, on days 1 and 3. Digital image analysis scripts were developed for segmentation of immunostained tissues. Staining intensities and localization (membrane vs. cytoplasm) of markers were assessed in >300,000 epithelial cells per section. Results: Epithelial transcriptional signatures were analyzed by RNA-sequencing of enteroids generated from jejunal crypts of Myo5b(G519R) and Myo5bΔIEC mice. Enteroids from both mice showed significant upregulation of Notch signaling mediators and the downregulation of tuft cell markers and mitochondrial metabolic genes compared to control enteroids. Immunostaining of tissue sections consistently demonstrated that functional Myo5b loss decreases tuft cell numbers and mitochondrial densities in crypt and villus epithelial cells. In the vehicle treated MVID mouse tissues, sodium transporters of enterocytes, such as Na+/H+ exchanger (NHE)3 and Na+-dependent glucose transporter (SGLT)1, were mislocalized from the apical membrane and colocalized with lysosomal markers similar to what is observed in MVID patient tissues. Compound-1 treatment increased the apical localization of NHE3 and SGLT1 both in Myo5b(G519R) and Myo5bΔIEC mouse small intestines, whereas Nirogacestat improved localizations only in Myo5bΔIEC but not Myo5b(G519R) mouse tissues. Conclusions: Functional MYO5B loss, caused by the point mutation at G519R or total knockout directly disrupts Notch signaling and energy metabolic pathways in the epithelial cells, resulting in differentiation defects and malnutrition. The distinct treatment effcacies between the two MVID mouse models suggest that the total loss and impaired function of MYO5B differentially affect epithelial cell functions. NIH R01DK128190, RC2DK118640. This is the full abstract presented at the American Physiology Summit 2024 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.

Colonic MYO5B Expression Retains Fecal Formation but not Weight Loss in OLFM4+ Stem Cell-Specific MYO5B Deletion in Mice

Background and aim: Microvillus inclusion disease (MVID) is a congenital disorder characterized by severe diarrhea and is caused by inactivating mutations in motor protein myosin Vb (MYO5B). Mice lacking MYO5B in intestinal and colonic epithelia ( Vil-CreERT2;Myo5bfl/fl) display an MVID phenotype characterized by significant weight loss, lack of solid feces, and brush border defects. However, previous models have not discerned if this diarrheal phenotype is due to MYO5B loss in the small intestine specifically or both in the small intestine and colon. Using the small intestinal stem cell specific Cre driver under Olfm4, we phenotyped the mice lacking MYO5B only in the small intestine, while normal MYO5B expression is maintained in the colon. Methods: Olfm4-GFP-CreERT2;R26R-LSL-tdTom and Myo5bfl/fl mice were crossbred to generate Olfm4-GFP-CreERT2;R26R-LSL-tdTom;Myo5bfl/fl (Olfm4DMYO5B) mice. Adult Olfm4DMYO5B mice and control littermates received tamoxifen injection at day 0. GI tissues were collected 5 and 7 days post tamoxifen. Results: Following Cre induction, Olfm4DMYO5B mice exhibited progressive weight loss starting at day 5 and on average lost 20.6% on day 7. At day 7, the small intestine contained clear luminal contents, similar to that of the previous MVID model mice. However, the colon of day 7 Olfm4DMYO5B mice contained small, solid feces. An elongation of PCNA+ crypts was identified on day 5 throughout the small intestine. Villus blunting and microvillus inclusions were prominent in the small intestinal enterocytes on day 7, but not on day 5. The day 7 Olfm4DMYO5B small intestine demonstrated a decrease in tuft cell numbers, suggesting differentiation deficits in the MYO5B-lacking stem cells. However, the colon of Olfm4DMYO5B mice showed intact morphology. Colonic crypt depth and tuft cell numbers at both days 5 and 7 were similar to those of control mice. Immunostaining confirmed the maintenance of MYO5B expression in the colon and progressive loss of MYO5B in the small intestine of Olfm4DMYO5B mice. At day 5, MYO5B was specifically lost in small intestinal crypts, and the loss of MYO5B expanded to the villus tips on day 7. These MYO5B-lacking cells were tdTom+, including enterocytes, goblet, and rare tuft cells. Following the loss of MYO5B, small intestinal enterocytes lacked proper apical localization of numerous transporters and enzymes, including Na+-glucose cotransporter 1 and dipeptidyl peptidase 4. Conclusion: Recently established Olfm4DMYO5B mice demonstrated that the specific MYO5B deletion in the small intestinal stem cells causes hyperproliferation and differentiation deficits. Colonic MYO5B function is suffcient for the formation of solid feces, however, these mice experience prominent weight loss, likely due to the MVID-like malabsorption in the small intestine. NIH R01DK128190, RC2DK118640. This is the full abstract presented at the American Physiology Summit 2024 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.

A286 MYO5B MUTATIONS AS A CAUSE OF CHOLESTASIS IN A CHILD WITHOUT MICROVILLOUS INCLUSION DISEASE

Abstract Background Biallelic Myosin Vb (MYO5B) mutations are identified in the majority of patients with microvillus inclusion disease (MVID) that requires life-long parenteral nutrition and eventually cholestatic liver disease. Biallelic MYO5B mutations have also been reported in a few children with predominant early-onset cholestatic liver disease. Aims To report on a patient with MYO5B mutations and progressive familial intrahepatic cholestasis PFIC-like phenotype and normal serum GGT without intestinal disease. Methods We describe a 2-year and 8-month-old girl, who is the first child of healthy non- consanguineous parents from the Phillipines with no family history of liver disease. She presented at the age of 10 months for evaluation of mild jaundice, which was noticed at the age of 6 months. Other symptoms included pruritus, slowly weight gain, intermittent pale stools and dark urine. Laboratory investigations revealed AST 99, ALT 87, GGT 27, conjugated bilirubin 13 and INR 0.8. Fasting abdominal ultrasound was normal apart from a possibly hypoplastic gallbladder and mild splenomegaly. Additional investigations revealed normal thyroid-stimulating hormone, cortisol, galactosemia screening and sweat chloride, but bile acids were elevated 391.6 umol/L. A liver biopsy was performed and a cholestasis genetic panel sent. The patient was started on ursodiol, hydroxyzine, rifampicin and vitamin D. Results Liver biopsy revealed moderate hepato-canalicular cholestasis, bile duct injury without prominent ductopenia, small caliber bile ducts with no features of obstructive cholangiopathy, mild periportal and pericentral fibrosis stage 1-2/ 4. Electron microscopy demonstrated cytoplasmic and canalicular bile accumulation. The bile canaliculi were variably distended by coarse granular bile. Immunohistochemical staining for BSEP and MDR3, showed canalicular staining in zones 1 and absence of staining in zones 2 and 3. Genetic analysis revealed one pathogenic variant and one variant of uncertain significance in MYO5B. No mutations in ABCB11 or ATP8B1 genes were identified. On follow-up, the jaundice improved, pruritus was managed with the medications described above. Her growth has improved and she is growing on the 25th centiles. She has not developed any gastrointestinal symptoms. Conclusions We present a patient with MYO5B mutations who presented with normal GGT cholestasis and a liver biopsy consistent with PFIC, without any evidence of intestinal disease. Because MYO5B mutations disrupt the intracellular trafficking of brush border proteins in the intestine, it has been assumed that it similarly disrupts the trafficking of the canalicular bile acid transporting and bilirubin transporting proteins in the liver. This case highlights the broad phenotype associated with MYO5B variants. Funding Agencies None

Approach to Congenital Diarrhea and Enteropathies (CODEs).

Congenital diarrhea and enteropathies (CODEs) constitute a group of rare genetic disorders characterized by severe diarrhea and malabsorption in the neonatal period or early infancy. Timely diagnosis and treatment is essential to prevent life-threatening complications, including dehydration, electrolyte imbalance, and malnutrition. This review offers a simplified approach to the diagnosis of CODEs, with a specific focus on microvillus inclusion disease (MVID), congenital tufting enteropathy (CTE), congenital chloride diarrhea (CLD), and congenital sodium diarrhea (CSD).Patients with CODEs typically present with severe watery or occasionally bloody diarrhea, steatorrhea, dehydration, poor growth, and developmental delay. Therefore, it is crucial to thoroughly evaluate infants with diarrhea to rule out infectious, allergic, or anatomical causes before considering CODEs as the underlying etiology. Diagnostic investigations for CODEs encompass various modalities, including stool tests, blood tests, immunological studies, endoscopy and biopsies for histology and electron microscopy, and next-generation sequencing (NGS). NGS plays a pivotal role in identifying the genetic mutations responsible for CODEs.Treatment options for CODEs are limited, often relying on total parenteral nutrition for hydration and nutritional support. In severe cases, intestinal transplantation may be considered. The long-term prognosis varies among specific CODEs, with some patients experiencing ongoing intestinal failure and associated complications. In conclusion, the early recognition and accurate diagnosis of CODEs are of paramount importance for implementing appropriate management strategies. Further research and advancements in genetic testing hold promise for enhancing diagnostic accuracy and exploring potential targeted therapies for these rare genetic disorders.

Patient-derived enteroids provide a platform for the development of therapeutic approaches in microvillus inclusion disease.

Microvillus inclusion disease (MVID), caused by loss-of-function mutations in the motor protein myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid/base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na+/H+ exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking antidiarrheal drug crofelemer dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. γ-Secretase inhibition with DAPT recovered apical brush border structure and functional Na+/H+ exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum/glucocorticoid-regulated kinase 2 (SGK2) and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID.

Lrig1+ small intestinal progenitor cells require MYO5B for proper crypt proliferation and enterocyte maturation

Microvillus inclusion disease (MVID) is a congenital disorder characterized by severe diarrhea. MVID is caused by inactivating mutations in myosin Vb (MYO5B), a motor protein. Mice lacking MYO5B in their intestinal epithelium have demonstrated the importance of MYO5B in enterocyte brush border formation and epithelial cell differentiation. Additionally, the proliferative zone of the intestinal crypt of MYO5B-deficient mice is elongated compared to controls. We hypothesize that the progenitor cell-specific Cre driver under Lrig1 demonstrates crypt-specific effects caused by MYO5B loss. Lrig1-CreERT2;R26R-YFP and Myo5bfl/fl mice were crossbred to generate Lrig1-CreERT2;R26R-YFP;MYO5B fl/fl (Lrig1 ΔMYO5B ) mice. Adult Lrig1 ΔMYO5B mice and control littermates received tamoxifen injection at day 0. GI tissues were collected 2.5, 3, 4, and 5 days after tamoxifen administration for histological characterization. Lrig1 ΔMYO5B mice began experiencing significant weight loss 4 days after tamoxifen injection and lost 20% of their starting body weight on day 5. At day 5, the Lrig1 ΔMYO5B mouse colon was devoid of solid feces and their small intestine contained clear luminal contents, together suggesting a watery diarrheal phenotype. Enterocytes of Lrig1DMYO5B mice on day 5 contained abnormal cytoplasmic accumulation of PAS staining. Fewer goblet cells were apparent in intestinal sections from day 3 and 5 Lrig1 ΔMYO5B mice compared to controls. Crypts were elongated starting at day 3. Similarly, the PCNA+ epithelial region was expanded in Lrig1 ΔMYO5B mice, even reaching into the villi at day 5. The time course of the PCNA+ region expansion corresponded to the wave of MYO5B-loss beginning at the base of crypts. MYO5B expression was diminished in the crypts at day 3 and nearly absent from the epithelium by day 5. Interestingly, expression of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), the rate-limited enzyme of ketogenesis that is important for stem cell function, was expanded in the crypt at day 3 and 5 after tamoxifen. Sodium/glucose cotransporter 1 (SGLT1), which is present along the apical surface of mature enterocytes, was progressively diminished and mis-localized away from the apical surface after tamoxifen administration. Apical sodium-dependent bile acid transporter (ASBT), found in the distal small intestine, demonstrated a similar mis-localization away from the apical membrane in enterocytes.We established a novel mouse model that demonstrates progressive MYO5B loss in the intestinal epithelium, starting at the base of the crypt and reaching villi tips over the course of 5 days following Cre induction. This Lrig1 ΔMYO5B strain will allow for lineage tracing studies to better understand the effect of MYO5B loss on intestinal epithelial differentiation and progenitor function. NSF GFRP, NIH R01DK128190 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.

Hepatic and ileal levels of targeted bile acids are altered in the murine myosin Vb knockout model of microvillus inclusion disease (MVID)

Background: Myosin Vb (Myo5b) is a molecular motor that traffics key cargo to the apical membrane of epithelial cells. Mutations in Myo5b that affect its function result in microvillus inclusion disease (MVID) in humans, which causes life-threatening diarrhea. Patients with MVID frequently present with liver cholestasis – a disorder resulting from reduced or blocked bile flow. Bile - primarily composed of cholesterol-derived bile acids (BAs) - is an important liver secretion that aids in the digestion of dietary fats and fat-soluble vitamins in the intestine. Primary BAs (such as cholic (CA) and chenodeoxycholic acid (CDA)) are synthesized in the liver and secreted into the intestine. In the intestine, primary bile acids are often converted by bacteria to secondary BAs (such as deoxycholic acid (DCA) and lithocholic acid (LCA)). Currently, the etiology of liver cholestasis and alterations in BA composition in the setting of loss of functional Myo5b is unknown. We hypothesized that Myo5b knockout (KO) mice would be unable to properly transport BAs in the liver and have lower levels of primary and secondary BAs in the intestine. Methods & Results: Liver and ileal contents was collected from Myo5b KO and littermate controls (n=3/genotype) and the tissue was weighed for normalization purposes. Each tissue sample was placed into individual homogenization tubes containing 100 mg of 1.4 mm ceramic beads and a volume equivalent to a tissue density of 200 mg/mL of ice-cold methanol. Tissue samples were then homogenized, tissue debris was pelleted by centrifugation, and the tissue sample supernatants were diluted in an Internal Standard solution prior to LC-MS/MS analysis. The BA (e.g., CA, β-MCA, CDCA, DCA, UDCA, LCA, GCA, GCDCA, GDCA, GUDCA, GLCA, TLCA, TCA, TCDCA, TDCA, TUDCA) content of the tissue sample extracts were quantified using a reverse-phase chromatography-tandem mass spectrometry (LC-MS/MS) method. Significant decreases in the concentrations of primary BAs (CA, β-MCA, GCA, TCA) and secondary BAs (DCA, TUDCA, TCDCA) were measured in the homogenized liver sample extracts collected from the Myo5b KO mice when compared to littermate control mice. Interestingly, we measured increased concentrations of primary BAs (CA and β-MCA) and one secondary BA (DCA) in the ileal tissue collected from the Myo5b KO mice when compared to littermate controls. We speculated that intestinal BAs could be accumulating due to loss of the apical BA transporter ASBT in the intestine. Immunostaining revealed loss of ASBT in the intestinal epithelium of adult mice with an intestinal specific deletion of Myo5b; indicating that Myo5b may regulate BAs levels via delivery of BAs transporters. Conclusion: Collectively, these data demonstrate the utility of our method for measuring the BA content of hepatic and GI tissue sample extracts in murine models of human disease, and the results suggest that the loss of functional Myo5b impairs liver BA production Financial support for this project was provided by an NIH-K01DK121869 and startup funds from MUSC (A Engevik). The Texas Children's Hospital Department of Pathology provides salary support to Texas Children's Microbiome Center-Metabolomics laboratory staff, and purchased the reagents, the consumables and durable supplies, and the LC-MS/MS equipment described. 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.

Generation of induced pluripotent stem cells (iPSCs) from a microvillus inclusion disease patient with a homozygous missense mutation in UNC45A

Mutations in UNC45A, a co-chaperone for myosins, were recently found causative of a syndrome combining cholestasis, diarrhea, loss of hearing and bone fragility. We generated induced pluripotent stem cells (iPSCs) from a patient with a homozygous missense mutation in UNC45A. Cells from this patient, which were reprogrammed using integration-free Sendaï virus, have normal karyotype, express pluripotency markers and are able to differentiate into the three germ cell layers.

Children with Microvillus Inclusion Disease in Oman

Children with Microvillus Inclusion Disease in Oman

Prikaz slučaja 30-godišnjeg muškarca sa megacistis-mikrokolon-intestinalnim hipoperistaltičkim sindromom sa de novo mutacijom gena ACTG2

Introduction: Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is a very rare genetic disorder of visceral motility of the gastrointestinal and genitourinary system. According to our knowledge, so far there has been no description of a patient with megacystis-microcolon-intestinal hypoperistalsis syndrome and chronic secretory diarrhea. Case report: We have presented a case report of a 30-year-old male with a genetically verified novel (de novo) mutation p.R257H in the smooth muscle actin (ACTG2) gene. At 8 months of age he was diagnosed with suspected Hirschsprung's disease, partial resection of the colon was performed. During the first year of his life, subtotal colectomy with cecorectal anastomosis was performed and, simultaneously, deteriorated emptying of the urinary bladder was confirmed. The patient was subjected to several unsuccessful abdominal and urological operations. At age of 18, the differential diagnosis was narrowed to micro-villus inclusion disease and congenital chloride diarrhea. The patient was tolerant to oral feeding all the time, where intermittent parenteral nutrition started only in adolescence. At the age of 26, due to urethral stenosis, perineal urethrostomy was performed. Since the age of 29, due to complications of the underlying disease the patient was administered chronic dialysis treatment, and a year later, genetic testing provided the definitive diagnosis of MMIHS. Therefore, combined kidney and intestinal transplantation was proposed. The patient continued the treatment with daily fluid and electrolyte compensation along with adequate parenteral nutrition through a triple-lumen central catheter. Conclusion: Timely genetic testing leads to avoiding repeated surgical interventions and numerous complications. Multivisceral transplantation represents a significant improvement in the treatment of patients with this syndrome.

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

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

Myosin Vb as a tumor suppressor gene in intestinal cancer.

Colorectal cancer causes >900,000 deaths every year and a deeper understanding of the molecular mechanisms underlying this disease will contribute to improve its clinical management and survival. Myosin Vb (MYO5B) regulates intracellular vesicle trafficking, and inactivation of this myosin disrupts the polarization and differentiation of intestinal epithelial cells causing microvillous inclusion disease (MVID), a rare congenital disorder characterized by intractable life-threatening diarrhea. Here, we show that the loss Myosin Vb interfered with the differentiation/polarization of colorectal cancer cells. Although modulation of Myosin Vb expression did not affect the proliferation of colon cancer cells, MYO5B inactivation increased their migration, invasion, and metastatic potential. Moreover, Myo5b inactivation in an intestine-specific knockout mouse model caused a >15-fold increase in the number of azoxymethane-initiated small intestinal tumors. Consistently, reduced expression of Myosin Vb in a cohort of 155 primary colorectal tumors was associated with shorter patient survival. In conclusion, we show here that loss of Myosin Vb reduces polarization/differentiation of colon cancer cells while enhancing their metastatic potential, demonstrating a tumor suppressor function for this myosin. Moreover, reduced expression of Myosin Vb in primary tumors identifies a subset of poor prognosis colorectal cancer patients that could benefit from more aggressive therapeutic regimens.

Compound Heterozygous MYO5B Mutation, a Cause of Infantile Cholestasis: A Case Report.

Infantile cholestasis is a common clinical problem in early infancy characterised by impairment in bile formation and/or flow. It requires prompt evaluation for underlying aetiology to initiate appropriate management. Although biliary atresia remains the most important aetiology, metabolic and monogenic disorders are increasingly identified with advances in diagnostic genetic testing. Progressive familial intrahepatic cholestasis disorders characterised by defects in biliary canalicular transport are among the most common monogenic disorders of cholestasis. Homozygous or compound heterozygous mutation in the Myosin 5B gene leading to a progressive familial intrahepatic cholestasis-like phenotype with or without intestinal features of microvillus inclusion disease is a relatively recently identified disorder. The incidence of these newer variants of progressive familial intrahepatic cholestasis is not yet known due to the paucity of studies. We report an uncommon cause of refractory cholestasis reported in a girl who presented with severe pruritus as the primary manifestation.

Fetal Bowel Abnormalities Suspected by Ultrasonography in Microvillus Inclusion Disease: Prevalence and Clinical Significance.

Microvillus inclusion disease (MVID) is a rare, inherited, congenital, diarrheal disorder that is invariably fatal if left untreated. Within days after birth, MVID presents as a life-threatening emergency characterized by severe dehydration, metabolic acidosis, and weight loss. Diagnosis is cumbersome and can take a long time. Whether MVID could be diagnosed before birth is not known. Anecdotal reports of MVID-associated fetal bowel abnormalities suspected by ultrasonography (that is, dilated bowel loops and polyhydramnios) have been published. These are believed to be rare, but their prevalence in MVID has not been investigated. Here, we have performed a comprehensive retrospective study of 117 published MVID cases spanning three decades. We find that fetal bowel abnormalities in MVID occurred in up to 60% of cases of MVID for which prenatal ultrasonography or pregnancy details were reported. Suspected fetal bowel abnormalities appeared in the third trimester of pregnancy and correlated with postnatal, early-onset diarrhea and case-fatality risk during infancy. Fetal bowel dilation correlated with MYO5B loss-of-function variants. In conclusion, MVID has already started during fetal life in a significant number of cases. Genetic testing for MVID-causing gene variants in cases where fetal bowel abnormalities are suspected by ultrasonography may allow for the prenatal diagnosis of MVID in a significant percentage of cases, enabling optimal preparation for neonatal intensive care.

Loss of Serum Glucocorticoid-Inducible Kinase 1 SGK1 Worsens Malabsorption and Diarrhea in Microvillus Inclusion Disease (MVID)

Microvillus inclusion disease (MVID), a lethal congenital diarrheal disease, results from loss of function mutations in the apical actin motor myosin VB (MYO5B). How loss of MYO5B leads to both malabsorption and fluid secretion is not well understood. Serum glucocorticoid-inducible kinase 1 (SGK1) regulates intestinal carbohydrate and ion transporters including cystic fibrosis transmembrane conductance regulator (CFTR). We hypothesized that loss of SGK1 could reduce CFTR fluid secretion and MVID diarrhea. Using CRISPR-Cas9 approaches, we generated R26CreER;MYO5Bf/f conditional single knockout (cMYO5BKO) and R26CreER;MYO5Bf/f;SGK1f/f double knockout (cSGK1/MYO5B-DKO) mice. Tamoxifen-treated cMYO5BKO mice resulted in characteristic features of human MVID including severe diarrhea, microvillus inclusions (MIs) in enterocytes, defective apical traffic, and depolarization of transporters. However, apical CFTR distribution was preserved in crypts and depolarized in villus enterocytes, and CFTR high expresser (CHE) cells were observed. cMYO5BKO mice displayed increased phosphorylation of SGK1, PDK1, and the PDK1 target PKCι in the intestine. Surprisingly, tamoxifen-treated cSGK1/MYO5B-DKO mice displayed more severe diarrhea than cMYO5BKO, with preservation of apical CFTR and CHE cells, greater fecal glucose and reduced SGLT1 and GLUT2 in the intestine. We conclude that loss of SGK1 worsens carbohydrate malabsorption and diarrhea in MVID.

UNC45A deficiency causes microvillus inclusion disease-like phenotype by impairing myosin VB-dependent apical trafficking.

Variants in the UNC45A cochaperone have been recently associated with a syndrome combining diarrhea, cholestasis, deafness, and bone fragility. Yet the mechanism underlying intestinal failure in UNC45A deficiency remains unclear. Here, biallelic variants in UNC45A were identified by next-generation sequencing in 6 patients with congenital diarrhea. Corroborating in silico prediction, variants either abolished UNC45A expression or altered protein conformation. Myosin VB was identified by mass spectrometry as client of the UNC45A chaperone and was found misfolded in UNC45AKO Caco-2 cells. In keeping with impaired myosin VB function, UNC45AKO Caco-2 cells showed abnormal epithelial morphogenesis that was restored by full-length UNC45A, but not by mutant alleles. Patients and UNC45AKO 3D organoids displayed altered luminal development and microvillus inclusions, while 2D cultures revealed Rab11 and apical transporter mislocalization as well as sparse and disorganized microvilli. All those features resembled the subcellular abnormalities observed in duodenal biopsies from patients with microvillus inclusion disease. Finally, microvillus inclusions and shortened microvilli were evidenced in enterocytes from unc45a-deficient zebrafish. Taken together, our results provide evidence that UNC45A plays an essential role in epithelial morphogenesis through its cochaperone function of myosin VB and that UNC45A loss causes a variant of microvillus inclusion disease.

Bridging Liver Transplantation in the Treatment of Intestinal Failure Associated Liver Disease in Infants-A Bridge Too Far?

Infants with intestinal failure associated liver disease (IFALD) requiring liver and bowel transplant have a high mortality on the transplant waiting list due to the scarcity of the size-matched donor organs. Bridging liver transplantation has been used to allow the children to grow to a reasonable size so that a combined liver and small bowel transplant could be performed in the future. We report on two children with irreversible intestinal failure (ultra-short bowel syndrome secondary to gastroschisis and microvillous inclusion disease) with IFALD who underwent bridging liver transplantation at our institution. Both patients made a good recovery from their initial surgery. One patient died 6 months following surgery from generalized sepsis, and the other patient survived in good condition to undergo a combined liver and small bowel transplant but died a few days post-transplant. In the current era of scarcity of donor organs, this raises an ethical dilemma for the team involved regarding appropriate utilisation of a scarce resource.