Microvillus Inclusion Disease Research Articles

A69 LONG-TERM EFFECTS OF TORKELSON SYNDROME

BackgroundTorkelson Syndrome is a rare, familial enteropathy with villous edema causing recurrent episodes of life threatening vomiting and secretory diarrhea in childhood, with children being well between episodes. The only description, of a Mennonite family from southern Alberta, suggests normal growth and no long term sequelae. Severe laboratory abnormalities common during bouts of diarrhea (hypoalbuminemia, neutropenia and electrolyte abnormalities) normalize in the asymptomatic phase except for decreased IgG subclasses. Typical findings from the duodenum and jejunum do not vary between acute episodes and when well. The pathophysiology is poorly understood, with acute episodes thought to be triggered by a non-specific infection in the setting of IgG2 deficiency.Apolipoprotein A-IV (APOA4) mutation has been documented in Torkelson syndrome. APOA4 is exclusively expressed in proximal small intestine villous enterocytes. It is thought to be involved in regulating pathways that control inflammation in enterocytes, resulting in decreased inflammation. It may prevent recruitment of leukocytes to inflamed intestinal enterocytes by suppressing P-selectin in the endothelial cells. Fully functioning APOA4 decreases secretion of IL-4 and TNF-a. In theory, abnormalities in APOA4, could result in a robust, unopposed inflammatory response in the small intestine.AimsReport possible long-term sequelae of Torkelson Syndrome.MethodsCase report.Results13 year old male with Torkelson syndrome presents with short stature and iron deficiency anemia responsive to oral iron. His weight is at the 0.1%ile, height at the 0.4%ile and BMI at the 1.6%ile.Life time hypoalbuminemia (20g/L) indicates chronic protein-losing enteropathy. Vitamin D is chronically low but responds to oral supplementation. IgA and IgM are normal, but IgG and its subclasses are decreased. Liver enzymes and electrolytes, including calcium and phosphate, outside of episodes of diarrhea are normal.Endoscopy shows prominent, thick villi, with fern-like appearance in the duodenum with normal stomach, esophagus, terminal ileum and colon. Biopsy results are consistent with previously described features of Torkelson syndrome with no additional abnormalities. MRE shows mild dilatation of the jejunum with hypertrophic conniventes with fat content. Abdominal ultrasound shows hepatomegaly with coarse echotexture and normal elastography. He has delayed bone age and low bone density.ConclusionsPatients with Torkelson syndrome, despite their recurrent episodes of life threatening vomiting and secretory diarrhea in childhood, were thought to have no long term sequelae including normal growth.Our patient, presents with chronic iron deficiency anemia, short stature, delayed bone age and density, and hepatomegaly of unknown significance. This case may broaden our knowledge of this poorly described condition to inform long term management of these patients.Funding AgenciesNone

MYO5B Gene Mutations: A Not Negligible Cause of Intrahepatic Cholestasis of Infancy With Normal Gamma-Glutamyl Transferase Phenotype.

Progressive familial intrahepatic cholestasis is an expanding group of autosomal recessive intrahepatic cholestatic disorders. Recently, next-generation sequencing allowed identifying new genes responsible for new specific disorders. Two biochemical phenotypes have been identified according to gamma-glutamyltransferase (GGT) activity. Mutations of the myosin 5B gene (MYO5B) are known to cause microvillus inclusion disease. Recently, different mutations in MYO5B gene have been reported in patients with low-GGT cholestasis. A multicenter retrospective and prospective study was conducted in 32 children with cryptogenic intrahepatic cholestasis. Clinical, biochemical, histological, and treatment data were analyzed in these patients. DNA from peripheral blood was extracted, and all patients were studied by whole exome sequencing followed by Sanger sequencing. Six patients out of 32 had mutations in the MYO5B gene. Of these six patients, the median age at disease onset was 0.8 years, and the median length of follow-up was 4.2 years. The most common signs were pruritus, poor growth, hepatomegaly, jaundice, and hypocholic stools. Two patients also showed intestinal involvement. Transaminases and conjugated bilirubin were moderately increased, serum bile acids elevated, and GGT persistently normal. At anti-Myo5B immunostaining, performed in liver biopsy of two patients, coarse granules were evident within the cytoplasm of hepatocytes while bile salt export pump was normally expressed at the canalicular membrane. Six variants in homozygosity or compound heterozygosity in the MYO5B gene were identified, and three of them have never been described before. All nucleotide alterations were located on the myosin motor domain except one missense variant found in the isoleucine-glutamine calmodulin-binding motif. We identified causative mutations in MYO5B in 18.7% of a selected cohort of patients with intrahepatic cholestasis confirming a relevant role for the MYO5B gene in low-GGT cholestasis.

Altered MYO5B Function Underlies Microvillus Inclusion Disease: Opportunities for Intervention at a Cellular Level.

Microvillus inclusion disease (MVID) is a congenital diarrheal disorder resulting in life-threatening secretory diarrhea in newborns. Inactivating and nonsense mutations in myosin Vb (MYO5B) have been identified in MVID patients. Work using patient tissues, cell lines, mice, and pigs has led to critical insights into the pathology of MVID and a better understanding of both apical trafficking in intestinal enterocytes and intestinal stem cell differentiation. These studies have demonstrated that loss of MYO5B or inactivating mutations lead to loss of apical sodium and water transporters, without loss of apical CFTR, accounting for the major pathology of the disease. In addition, loss of MYO5B expression induces the formation of microvillus inclusions through apical bulk endocytosis that utilizes dynamin and PACSIN2 and recruits tight junction proteins to the sites of bulk endosome formation. Importantly, formation of microvillus inclusions is not required for the induction of diarrhea. Recent investigations have demonstrated that administration of lysophosphatidic acid (LPA) can partially reestablish apical ion transporters in enterocytes of MYO5B KO mice. In addition, further studies have shown that MYO5B loss induces an imbalance in Wnt/Notch signaling pathways that can lead to alterations in enterocyte maturation and tuft cell lineage differentiation. Inhibition of Notch signaling leads to improvements in those cell differentiation deficits. These studies demonstrate that directed strategies through LPA receptor activation and Notch inhibition can bypass the inhibitory effects of MYO5B loss. Thus, effective strategies may be successful in MVID patients and other congenital diarrhea syndromes to reestablish proper apical membrane absorption of sodium and water in enterocytes and ameliorate life-threatening congenital diarrhea.

MYO5B mutation causing cholestasis in the absence of Microvillous Inclusion Disease: a rare case report

Background and Aim: Gene mutations known to cause Progressive Familial Intrahepatic Cholestasis (PFIC) 1, 2 and 3 have been long known. Newer variants, labelled PFIC 4, 5 and 6 have been described in recent literature. Myosin 5B (Myo5B) mutation is known to cause Microvillous Inclusion Disease (MVID) and has recently also been implicated in PFIC 6, a rare entity with only few documented case reports. We describe one such case report of a young girl presenting with cholestasis with normal serum Gamma-Glutamyl Transferase (GGT) activity, without MVID, diagnosed to have PFIC 6.

A Functional Relationship Between UNC45A and MYO5B Connects Two Rare Diseases With Shared Enteropathy.

Background & AimsUNC45A is a myosin (co-)chaperone, and mutations in the UNC45A gene were recently identified in osteo-oto-hepato-enteric (O2HE) syndrome patients presenting with congenital diarrhea and intrahepatic cholestasis. Congenital diarrhea and intrahepatic cholestasis are also the prime symptoms in patients with microvillus inclusion disease (MVID) and mutations in MYO5B, encoding the recycling endosome–associated myosin Vb. The aim of this study was to determine whether UNC45A and myosin Vb are functionally linked.MethodsCRISPR-Cas9 gene editing and site-directed mutagenesis were performed with intestinal epithelial and hepatocellular cell lines, followed by Western blotting, quantitative polymerase chain reaction, and scanning electron and/or confocal fluorescence microscopy to determine the relationship between (mutants of) UNC45A and myosin Vb.ResultsUNC45A depletion in intestinal and hepatic cells reduced myosin Vb protein expression, and in intestinal epithelial cells, it affected 2 myosin Vb-dependent processes that underlie MVID pathogenesis: rat sarcoma-associated binding protein (RAB)11A-positve recycling endosome positioning and microvilli development. Reintroduction of UNC45A in UNC45A-depleted cells restored myosin Vb expression, and reintroduction of UNC45A or myosin Vb, but not the O2HE patient UNC45A-c.1268T>A variant, restored recycling endosome positioning and microvilli development. The O2HE patient-associated p.V423D substitution, encoded by the UNC45A-c.1268T>A variant, impaired UNC45A protein stability but as such not the ability of UNC45A to promote myosin Vb expression and microvilli development.ConclusionsA functional relationship exists between UNC45A and myosin Vb, thereby connecting 2 rare congenital diseases with overlapping enteropathy at the molecular level. Protein instability rather than functional impairment underlies the pathogenicity of the O2HE syndrome–associated UNC45A-p.V423D mutation.

Newer variants of progressive familial intrahepatic cholestasis.

Progressive familial intrahepatic cholestasis (PFIC) is a heterogeneous group of disorders characterized by defects in bile secretion and presentation with intrahepatic cholestasis in infancy or childhood. The most common types include PFIC 1 (deficiency of FIC1 protein, ATP8B1 gene mutation), PFIC 2 (bile salt export pump deficiency, ABCB11 gene mutation), and PFIC 3 (multidrug resistance protein-3 deficiency, ABCB4 gene mutation). Mutational analysis of subjects with normal gamma-glutamyl transferase cholestasis of unknown etiology has led to the identification of newer variants of PFIC, known as PFIC 4, 5, and MYO5B related (sometimes known as PFIC 6). PFIC 4 is caused by the loss of function of tight junction protein 2 (TJP2) and PFIC 5 is due to NR1H4 mutation causing Farnesoid X receptor deficiency. MYO5B gene mutation causes microvillous inclusion disease (MVID) and is also associated with isolated cholestasis. Children with TJP2 related cholestasis (PFIC-4) have a variable spectrum of presentation. Some have a self-limiting disease, while others have progressive liver disease with an increased risk of hepatocellular carcinoma. Hence, frequent surveillance for hepatocellular carcinoma is recommended from infancy. PFIC-5 patients usually have rapidly progressive liver disease with early onset coagulopathy, high alpha-fetoprotein and ultimately require a liver transplant. Subjects with MYO5 B-related disease can present with isolated cholestasis or cholestasis with intractable diarrhea (MVID). These children are at risk of worsening cholestasis post intestinal transplant (IT) for MVID, hence combined intestinal and liver transplant or IT with biliary diversion is preferred. Immunohistochemistry can differentiate most of the variants of PFIC but confirmation requires genetic analysis.

MYO5B-associated diseases: Novel liver-related variants and genotype-phenotype correlation.

Biallelic pathogenic variants in MYO5B cause microvillus inclusion disease (MVID), or familial intrahepatic cholestasis (FIC). The reported FIC patients are scarce and so the genotype-phenotype correlation has not been fully characterised. This study aimed to report more MYO5B-associated FIC patients and correlate genotypes to phenotypes in more detail. The phenotype and genetic data of 12 newly diagnosed MYO5B-associated (including 11 FIC) patients, as well as 118 previously reported patients with available genotypes, were summarised. Only patients with biallelic MYO5B variants were enrolled. Nonsense, frameshift, canonical splice sites, initiation codon loss, and single exon or multiexon deletion were defined as null MYO5B variants. Phenotypically, 50 were isolated MVID, 47 involved both liver and intestine (combined), and 33 were isolated FIC (9 persistent, 15 recurrent, 3 transient, and 6 un-sub-classified) patients. The severity of intestinal manifestation was positively correlated to an increased number of null variants (ρ=0.299, P=.001). All FIC patients carried at least one non-null variant, and the severity of cholestasis was correlated to the presence of a null variant (ρ=0.420, P=.029). The proportion of FIC patients (16/29, 55%) harbouring missense/in-frame variants affecting the non-motor regions of MYO5B was significantly higher than that of MVID (3/25, 12%, P=.001) and combined patients (3/31, 10%, P=.000). 10 of the 29 FIC patients harboured missense/in-frame variants at the IQ motifs comparing to none in the 56 MVID and combined patients (P=.000). The phenotype of MYO5B deficiency was associated with MYO5B genotypes, the nullity or the domain affected.

Novel MYO5B mutation in microvillous inclusion disease of Syrian ancestry.

Microvillus inclusion disease (MVID) is a rare autosomal recessive condition characterized by a lack of microvilli on the surface of enterocytes, resulting in severe, life-threatening diarrhea that could lead to mortality within the first year of life. We identify two unrelated families, each with one child presenting with severe MVID from birth. Using trio whole-exome sequencing, we observed that the two families share a novel nonsense variant (Glu1589*) in the MYO5B gene, a type Vb myosin motor protein in which rare damaging mutations were previously described to cause MVID. This founder mutation was very rare in public databases and is likely specific to patients of Syrian ancestry. We present a detailed account of both patients’ clinical histories to fully characterize the effect of this variant and expand the genotype–phenotype databases for MVID patients from the Middle East.

Monogenic mutations in four cases of neonatal-onset watery diarrhea and a mutation review in East Asia

BackgroundInfants with neonatal-onset diarrhea present with intractable diarrhea in the first few weeks of life. A monogenic mutation is one of the disease etiologies and the use of next-generation sequencing (NGS) has made it possible to screen patients for their mutations.Main bodyWe retrospectively reviewed the clinical data of four children from unrelated families, who presented with neonatal-onset, chronic, watery, non-bloody diarrhea. After genetic whole-exome sequencing, novel mutations were identified in the EPCAM gene of two children. Congenital chloride diarrhea was diagnosed in one case, which was associated with an SLC26A3 mutation, in which the patient presented with watery diarrhea, malnutrition, and hypochloremic alkalosis. Patient 4 was diagnosed with microvillus inclusion disease and possessed novel compound heterozygous mutations in the MYO5B gene. A review of the genetic variants of SLC26A3 reported in East Asia revealed that c.269_270 dupAA (p.G91Kfs*3) is the most frequent SLC26A3 mutation in China, compared with c.2063-1 G > T in Japan and Korea. EPCAM and MYO5B genetic variants were only sporadically reported in East Asia.ConclusionThis study expands our knowledge of the clinical manifestations and molecular genetics of neonatal-onset watery diarrhea. Early diagnosis could be achieved by genomic analysis in those infants whose histology features are not typical. The discovery of four novel mutations in the EPCAM gene and two novel mutations in the MYO5B gene provides further etiological evidence for the association of genetic mutations with neonatal-onset diarrhea. To date, c.269_270 dupAA is the most frequent SLC26A3 mutation in China.

Risk and Clinical Significance of Idiopathic Preterm Birth in Microvillus Inclusion Disease.

Microvillus inclusion disease (MVID) is a rare enteropathy caused by mutations in the MYO5B or STX3 gene. MVID is a disease that is difficult to manage with clinical heterogeneity. Therefore, knowledge about factors influencing MVID morbidity and mortality is urgently needed. Triggered by a recent study that reported a high percentage of preterm births in twelve cases of MVID, we have conducted a comprehensive retrospective study involving 88 cases of MVID with reported gestational ages. We found that moderate to late preterm birth occurred in more than half of all cases, and this was particularly prominent in MYO5B-associated MVID. Preterm birth in MVID counterintuitively correlated with higher birth weight percentiles, and correlated with higher stool outputs and a significantly shorter average survival time. Data from this study thus demonstrate an increased risk of preterm birth in MYO5B-associated MVID, with a clinical impact on morbidity and mortality. Adverse effects associated with preterm birth should be taken into account in the care of children diagnosed with MVID. Documentation of gestational age may contribute to a better prognostic risk assessment in MVID.

Cell differentiation is disrupted by MYO5B loss through Wnt/Notch imbalance.

Functional loss of myosin Vb (MYO5B) induces a variety of deficits in intestinal epithelial cell function and causes a congenital diarrheal disorder, microvillus inclusion disease (MVID). The impact of MYO5B loss on differentiated cell lineage choice has not been investigated. We quantified the populations of differentiated epithelial cells in tamoxifen-induced, epithelial cell–specific MYO5B-knockout (VilCreERT2 Myo5bfl/fl) mice utilizing digital image analysis. Consistent with our RNA-sequencing data, MYO5B loss induced a reduction in tuft cells in vivo and in organoid cultures. Paneth cells were significantly increased by MYO5B deficiency along with expansion of the progenitor cell zone. We further investigated the effect of lysophosphatidic acid (LPA) signaling on epithelial cell differentiation. Intraperitoneal LPA significantly increased tuft cell populations in both control and MYO5B-knockout mice. Transcripts for Wnt ligands were significantly downregulated by MYO5B loss in intestinal epithelial cells, whereas Notch signaling molecules were unchanged. Additionally, treatment with the Notch inhibitor dibenzazepine (DBZ) restored the populations of secretory cells, suggesting that the Notch pathway is maintained in MYO5B-deficient intestine. MYO5B loss likely impairs progenitor cell differentiation in the small intestine in vivo and in vitro, partially mediated by Wnt/Notch imbalance. Notch inhibition and/or LPA treatment may represent an effective therapeutic approach for treatment of MVID.

Antenatal diagnosis of microvillus inclusion disease

Microvillus inclusion disease is a rare autosomal recessive disorder due to defective apical surface of the enterocytes presenting with severe watery diarrhea starting at birth. We describe a female infant who had antenatal diagnosis of microvillus inclusion disease. At 36th gestational week of a 32-year-old woman ultrasound examination revealed dilatation of fetal sigmoid colon. The amniotic fluid level was normal. An amniocentesis was performed to rule out congenital sodium and chloride diarrhea in the prenatal period. The patient didn't prefer to undergo genetic tests. In conclusion, prenatal ultrasonographic identification of dilated bowel loops without polyhydramnios suggests differential diagnosis of microvillus inclusion disease in addition to congenital chloride diarrhea, jejunoileal atresia, volvulus, meconium ileus, Hirschsprung disease, enteric duplications, anorectal atresia.

Congenital Diarrhea and Enteropathies in Infants: Approach to Diagnosis.

Congenital diarrhea and enteropathies (CODEs) are monogenic disorders causing early onset of intractable diarrhea. Their diagnosis and management are challenging. With the availability of commercial next generation genetic testing, we are now better able to classify and manage these disorders. The authors present their experience with 4 cases. Two patients had congenital tufting enteropathy (CTE) and 1 case each of microvillous inclusion disease (MVID) and trichohepatoenteric syndrome (THES). Age at onset varied from 3 to 38 d of life. Light microscopy and electron microscopy of duodenal and rectal endoscopic biopsies were consistent with the diagnosis. Genetic evaluation was possible in 3 cases indicating causative mutations. Two children (CTE and MVID) were alive at last follow-up. The authors suggest a stepwise approach to the diagnosis and management of these disorders in the Indian context.

Genetic Disorders of Bile Acid Transport.

Genetic Disorders of Bile Acid Transport.

Pathogenic STX3 variants affecting the retinal and intestinal transcripts cause an early-onset severe retinal dystrophy in microvillus inclusion disease subjects

Biallelic STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic—intestinal and retinal—disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.

Loss of Myosin Vb Results in Decreased Apical P‐Glycoprotein in vivo and in vitro

Background & Aims P-glycoprotein (p-gp), also known as multidrug resistance protein 1, is a xenobiotic efflux pump. In the small intestine, p-gp is localized to the apical membrane of enterocytes. Changes in p-gp expression is linked to inflammatory bowel diseases (IBD) and impairment of drug response in cancer. Regulating the trafficking of p-gp to the plasma membrane represents a plausible approach to improve therapy for cancer treatment. At present, the trafficking mechanism responsible for delivery of p-gp to the apical membrane of epithelial cells is unclear. Myosin Vb (Myo5b) is a molecular motor that is crucial for the proper transport of vesicles and proteins to the apical membrane of epithelial cells. Loss of functional Myo5b results in a congenital diarrheal disorder termed Microvillus Inclusion Disease (MVID). Individuals with MVID present with severe, unremitting diarrhea requiring parenteral nutrition and eventual small bowel transplantation. Studies using animal models of MVID and MVID patient tissue have revealed that loss of Myo5b results in a dramatic decrease in key transporters on the apical membrane of enterocytes. Therefore, we investigated whether proper localization of p-glycoprotein was impeded by loss of Myo5b. Methods Intestinal tissue was analyzed from neonatal control and germline Myo5b knockout (Myo5b KO) mice. Additionally, a tamoxifen inducible, intestinal specific mouse model of loss of Myo5b was also used (VillinCreERT2;Myo5bflox/flox). Adult Myo5bflox/flox mice treated with tamoxifen were used as controls for VillinCreERT2;Myo5bflox/flox mice. Intestinal organoids were generated from VillinCreERT2;Myo5bflox/flox mice and treated with either ethanol as a control or 4 hydroxy tamoxifen to induce loss of Myo5b. Results Immunostaining of gamma actin staining was performed to delineate the actin rich microvilli and identify the apical membrane. In control mice, p-gp was restricted to the apical membrane and co-localized with gamma actin as expected. In germline Myo5b KO mice, p-gp had increased immunofluorescence intensity and was observed below the apical membrane of small intestinal enterocytes. Lamp1 immunostaining also revealed that subapical p-gp was surrounded by lamp1 positive lysosomes in Myo5b KO mice. The association of lysosomes with p-gp suggests that improper localization of p-gp resulting from loss of Myo5b may promote lysosomal degradation of p-gp. Adult Myo5bflox/flox control mice treated with tamoxifen showed normal distribution of p-gp on the apical membrane of small intestinal enterocytes demonstrating co-localization with gamma actin. In contrast, tamoxifen induced VillinCreERT2;Myo5bflox/flox mice showed no p-gp at the apical brush border and no overlap with gamma actin immunostaining. Tamoxifen induced VillinCreERT2;Myo5bflox/flox mice displayed a large accumulation of p-gp below the apical membrane. Complementing our in vivo data, in vitro induction of Myo5b loss using 4 hydroxy tamoxifen resulted in a similar subapical localization of p-gp in induced VillinCreERT2;Myo5bflox/flox intestinal organoids compared to ethanol treated organoids. Conclusions Loss of Myo5b results in improper localization of p-gp. Collectively, these data suggest that Myo5b is necessary for the proper trafficking of p-gp to the apical membrane of intestinal cells.

Functional MYO5B Loss Disrupts Stem Cell Function and Differentiation of Secretory Cell Lineage

Background and Aims Differentiation of intestinal epithelial cells involves cell division inhibition, cell lineage choice, and brush border elaboration. Functional myosin Vb (MYO5B) loss causes microvillus inclusion disease (MVID) and induces a variety of deficits in enterocyte function. However, the impact of MYO5B loss on differentiated cell lineage choice had not been investigated. Since intestinal progenitor cells predominantly express MYO5B, we hypothesize that MYO5B deficiency alters stem/progenitor cell function and affects proper cell differentiation. Methods Tamoxifen-induced epithelial-specific MYO5B knockout (VilCreERT2; Myo5bflox/flox) mice were used for immunohistochemical analysis of intestinal tissues, organoid generation from crypts, and RNA-sequencing of isolated epithelial cells. We quantified the population of secretory cell lineages in utilizing whole slide imaging and digital image analysis tools. Neonatal pig intestinal organoids, possessing the Navajo mutation MYO5B(P663L), were analyzed as another MVID model that more closely represents human MVID pathology. Results Consistent with our RNA-sequencing data in epithelial cells that demonstrated decreases in Pou2f3, Spib, Dclk1, Chat, and Pgst1, MYO5B loss induced an 80% reduction in DCLK1-positive tuft cells. The frequency of TFF3-producing goblet cells was also significantly decreased. However, the Paneth cell lineage was increased by MYO5B deficiency along with expansion of the progenitor cell zone that express a Notch-dependent stem cell marker, olfactomedin (OLFM)4, and monocarboxylate transporter (MCT)1. No difference was detected in chromogranin-A-expressing enteroendocrine cells. mRNA expression indicated an increase in Notch receptor and decreases in Wnt family ligands in MYO5B deficient intestine, which may explain the alteration of cell lineages by MYO5B loss. We further investigated the effect of lysophosphatidic acid (LPA) receptor activation on epithelial sensory cell differentiation. Intraperitoneal, but not gavaged, LPA significantly increased tuft cell populations both in control and MYO5B knockout mice without alteration of mRNA expression of tuft cell-specific transcription factors. LPA did not affect hyperproliferative crypts in MYO5B deficient mice. In mouse intestinal organoids, the frequency of tuft cells was significantly decreased by 4-hydroxy-tamoxifen-induced MYO5B loss to values corresponding to undifferentiated enteroids. Intestinal organoids that were generated from MYO5B(P663L) pig jejunum had grater organoid forming rate and more proliferative cell population compared to age-matched wild type pig organoids. Differentiation of secretory cell lineages were inhibited in MYO5B(P663L) organoids similarly to the mouse model. Conclusions Functional MYO5B loss disrupts stem cell characteristics and proper differentiation in the small intestine likely through imbalance of Notch/Wnt signaling. Systemic LPA administration specifically activates tuft cell differentiation independent from MYO5B-mediated signaling pathway.

Advanced Microscopy for Liver and Gut Ultrastructural Pathology in Patients with MVID and PFIC Caused by MYO5B Mutations.

Mutations in the actin motor protein myosinVb (myo5b) cause aberrant apical cargo transport and the congenital enteropathy microvillus inclusion disease (MVID). Recently, missense mutations in myo5b were also associated with progressive familial intrahepatic cholestasis (MYO5B-PFIC). Here, we thoroughly characterized the ultrastructural and immuno-cytochemical phenotype of hepatocytes and duodenal enterocytes from a unique case of an adult MYO5B-PFIC patient who showed constant hepatopathy but only periodic enteric symptoms. Selected data from two other patients supported the findings. Advanced methods such as cryo-fixation, freeze-substitution, immuno-gold labeling, electron tomography and immuno-fluorescence microscopy complemented the standard procedures. Liver biopsies showed mislocalization of Rab11 and bile canalicular membrane proteins. Rab11-positive vesicles clustered around bile canaliculi and resembled subapical clusters of aberrant recycling endosomes in enterocytes from MVID patients. The adult patient studied in detail showed a severe, MVID-specific enterocyte phenotype, despite only a mild clinical intestinal presentation. This included mislocalization of numerous proteins essential for apical cargo transport and morphological alterations. We characterized the heterogeneous population of large catabolic organelles regarding their complex ultrastructure and differential distribution of autophagic and lysosomal marker proteins. Finally, we generated duodenal organoids/enteroids from biopsies that recapitulated all MVID hallmarks, demonstrating the potential of this disease model for personalized medicine.

NGS Gene Panel Analysis Revealed Novel Mutations in Patients with Rare Congenital Diarrheal Disorders.

Congenital diarrheal disorders (CDDs) are early-onset enteropathies generally inherited as autosomal recessive traits. Most patients with CDDs require rapid diagnosis as they need immediate and specific therapy to avoid a poor prognosis, but their clinical picture is often overlapping with a myriad of nongenetic diarrheal diseases. We developed a next-generation sequencing (NGS) panel for the analysis of 92 CDD-related genes, by which we analyzed patients suspect for CDD, among which were (i) three patients with sucrose-isomaltase deficiency; (ii) four patients with microvillous inclusion disease; (iii) five patients with congenital tufting enteropathy; (iv) eight patients with glucose-galactose malabsorption; (v) five patients with congenital chloride diarrhea. In all cases, we identified the mutations in the disease-gene, among which were several novel mutations for which we defined pathogenicity using a combination of bioinformatic tools. Although CDDs are rare, all together, they have an incidence of about 1%. Considering that the clinical picture of these disorders is often confusing, a CDD-related multigene NGS panel contributes to unequivocal and rapid diagnosis, which also reduces the need for invasive procedures.

Congenital Diarrhea and Cholestatic Liver Disease: Phenotypic Spectrum Associated with MYO5B Mutations.

Myosin Vb (MYO5B) is a motor protein that facilitates protein trafficking and recycling in polarized cells by RAB11- and RAB8-dependent mechanisms. Biallelic MYO5B mutations are identified in the majority of patients with microvillus inclusion disease (MVID). MVID is an intractable diarrhea of infantile onset with characteristic histopathologic findings that requires life-long parenteral nutrition or intestinal transplantation. A large number of such patients eventually develop cholestatic liver disease. Bi-allelic MYO5B mutations are also identified in a subset of patients with predominant early-onset cholestatic liver disease. We present here the compilation of 114 patients with disease-causing MYO5B genotypes, including 44 novel patients as well as 35 novel MYO5B mutations, and an analysis of MYO5B mutations with regard to functional consequences. Our data support the concept that (1) a complete lack of MYO5B protein or early MYO5B truncation causes predominant intestinal disease (MYO5B-MVID), (2) the expression of full-length mutant MYO5B proteins with residual function causes predominant cholestatic liver disease (MYO5B-PFIC), and (3) the expression of mutant MYO5B proteins without residual function causes both intestinal and hepatic disease (MYO5B-MIXED). Genotype-phenotype data are deposited in the existing open MYO5B database in order to improve disease diagnosis, prognosis, and genetic counseling.