Uncovering the Relationship Between Genes and Phenotypes Beyond the Gut in Microvillus Inclusion Disease.

Microvillus inclusion disease (MVID) is a rare condition that is present from birth and affects the digestive system. People with MVID experience severe diarrhea that is difficult to control, cannot absorb dietary nutrients, and struggle to grow and thrive. In addition, diverse clinical manifestations, some of which are life-threatening, have been reported in cases of MVID. MVID can be caused by variants in the MYO5B, STX3, STXBP2, or UNC45A gene. These genes produce proteins that have been functionally linked to each other in intestinal epithelial cells. MVID associated with STXBP2 variants presents in a subset of patients diagnosed with familial hemophagocytic lymphohistiocytosis type 5. MVID associated with UNC45A variants presents in most patients diagnosed with osteo-oto-hepato-enteric syndrome. Furthermore, variants in MYO5B or STX3 can also cause other diseases that are characterized by phenotypes that can co-occur in subsets of patients diagnosed with MVID. Recent studies involving clinical data and experiments with cells and animals revealed connections between specific phenotypes occurring outside of the digestive system and the type of gene variants that cause MVID. Here, we have reviewed these patterns and correlations, which are expected to be valuable for healthcare professionals in managing the disease and providing personalized care for patients and their families.

Malabsorption Spectrums in India.

Among the causes of malabsorption, tropical sprue is one of the leading cause.Several reports indicating that celiac disease, now being recognised more frequently. MATERIAL: 94 patients, aged more than 12 years, presenting with Chronic diarrhoea and malabsorption syndrome were analyzed by clinical presentation, endoscopic and histopathological examination.The spectrum of disease in these patients and features differentiating celiac disease and tropical sprue are reported here. OBSERVATION: Most common cause was Celiac Disease (65%), followed by Tropical Sprue (21%), common variable immunodeficiency (2%), lymphangiectasia (1%), idiopathic (3%). Patients with celiac disease were younger,having anemia, scalloping of folds,moderate or severe villous atrophy, crypt hyperplasia, diffuse epithelial damage. Patients with tropical sprue were older and more often normal duodenal epithelium. CONCLUSION: Malabsorption, a disease which is often missed and not recognised by clinicians. A meticulous search for diagnosis is required.

Exocrine pancreatic insufficiency related fat malabsorption and its association with autonomic neuropathy in Asian Indians with type 2 diabetes mellitus.

BACKGROUND AND AIMS: We aimed to estimate the prevalence of exocrine pancreatic insufficiency (EPI) related fat malabsorption & to correlate it with measures of autonomic neuropathy in patients with T2DM from India. METHODS: Patients with T2DM (cases; n = 118) and normo-glycaemic individuals (controls; n = 82) underwent anthropometry and biochemical evaluation at baseline. The 72-hours fecal fat excretion was estimated by the Van de Kamer's titration method. Autonomic neuropathy was evaluated using an automated analyzer. RESULTS: The prevalence of EPI related fat malabsorption in cases was 45% (n = 53; 72 hours mean fecal fat level = 22.7 ± 5.6 g/day). Dysfunctions in the parasympathetic nervous system (PNS; 86.7%; p < 0.05), sympathetic nervous system (SNS; 92.4%; p < 0.05), and both; PNS + SNS (83.1%; p < 0.05) were significant. Amongst measures of PNS dysfunction, there was a significantly higher percentage of abnormal expiration: inspiration ratio (45.3%) and the 30:15 ratio (84.9%) (p < 0.05) in patients with T2DM and EPI related fat malabsorption. CONCLUSION: In this cross-sectional cohort of Asian Indian patients with T2DM (n = 118), EPI related fat malabsorption correlates significantly with autonomic dysfunction in patients with T2DM. However, these preliminary data need to confirmed in trials with more robust design.

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.

Aberrant Epithelial Differentiation Contributes to Pathogenesis in a Murine Model of Congenital Tufting Enteropathy.

BACKGROUND & AIMS: Congenital tufting enteropathy (CTE) is an intractable diarrheal disease of infancy caused by mutations of epithelial cell adhesion molecule (EpCAM). The cellular and molecular basis of CTE pathology has been elusive. We hypothesized that the loss of EpCAM in CTE results in altered lineage differentiation and defects in absorptive enterocytes thereby contributing to CTE pathogenesis. METHODS: Intestine and colon from mice expressing a CTE-associated mutant form of EpCAM (mutant mice) were evaluated for specific markers by quantitative real-time polymerase chain reaction, Western blotting, and immunostaining. Body weight, blood glucose, and intestinal enzyme activity were also investigated. Enteroids derived from mutant mice were used to assess whether the decreased census of major secretory cells could be rescued. RESULTS: Mutant mice exhibited alterations in brush-border ultrastructure, function, disaccharidase activity, and glucose absorption, potentially contributing to nutrient malabsorption and impaired weight gain. Altered cell differentiation in mutant mice led to decreased enteroendocrine cells and increased numbers of nonsecretory cells, though the hypertrophied absorptive enterocytes lacked key features, causing brush border malfunction. Further, treatment with the Notch signaling inhibitor, DAPT, increased the numbers of major secretory cell types in mutant enteroids (graphical abstract 1). CONCLUSIONS: Alterations in intestinal epithelial cell differentiation in mutant mice favor an increase in absorptive cells at the expense of major secretory cells. Although the proportion of absorptive enterocytes is increased, they lack key functional properties. We conclude that these effects underlie pathogenic features of CTE such as malabsorption and diarrhea, and ultimately the failure to thrive seen in patients.

Expanding the clinical spectrum in trichohepatoenteric syndrome.

Trichohepatoenteric syndrome (THES) is a very rare autosomal recessive genetic disorder, which is characterized by intractable diarrhea during infancy, dysmorphic features, immunodeficiency, and a failure to thrive. There are still significant difficulties for patients and clinicians in terms of the management of THES, even though its molecular basis has been uncovered in the last decade. In this article, we have presented two cases relating to siblings that have been diagnosed with the condition. Concerning one of the patients, we described a novel variation (c.2114 + 5G > A) in the TTC37 gene and a mild clinical course; meanwhile, the other one was clinically diagnosed with THES at 17 years of age, but they had seizures and died suddenly. These cases expand the spectrum of clinical findings in relation to THES.

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.

The role of histopathology in the diagnosis and management of coeliac disease and other malabsorptive conditions.

Most absorption of nutrients takes place in the proximal small intestine, and the most common disorders leading to malabsorption are associated with a morphological abnormality in the duodenal mucosa that is appreciable in histological sections of biopsy specimens. Coeliac disease is the most well-known example, causing intraepithelial lymphocytosis, inflammation and villous atrophy in the duodenum. Remarkably similar inflammatory changes can be induced by other processes, including medications, e.g. angiotensin II receptor blockers and immune checkpoint inhibitors, immune dysregulation disorders, e.g. common variable immunodeficiency and autoimmune enteropathy, infections, collagenous sprue, and tropical sprue. However, there are often subtle histological differences from coeliac disease in the type of inflammatory infiltrate, the presence of crypt apoptosis, and the extent and type of inflammation beyond the duodenum. The clinical setting and serological investigation usually allow diagnostic separation, but some cases remain challenging. Histopathology is also important in assessing the response to treatment, such as the change in villous architecture caused by a gluten-free diet, or the response to cessation of a potentially causative medication. This review examines the practical role that histopathology of duodenal biopsy specimens plays in the assessment and management of inflammatory malabsorptive processes of the proximal small intestine, with a particular emphasis on coeliac disease.

Bile acid diarrhoea: Current and potential methods of diagnosis.

Chronic diarrhoea is common and mostly due to diarrhoea predominant irritable bowel syndrome. Diarrhoea predominant irritable bowel syndrome affects about 11% of the population; however, up to a third of these patients actually have bile acid diarrhoea. There are, therefore, more than one million sufferers of bile acid diarrhoea in the UK. Bile acid diarrhoea is caused by small bowel malabsorption of bile acids and the increased bile acids in the large intestine cause diarrhoea. Once diagnosed, the treatment of bile acid diarrhoea is simple and effective. Bile acid diarrhoea , however, is often not diagnosed because of a lack of easily available and reliable diagnostic methods. In the United Kingdom, the radiolabelled 23-seleno-25-homotaurocholic acid test is the gold-standard method of diagnosis. 23-seleno-25-homotaurocholic acid test, however, is expensive, inconvenient to the patient, involves radiation exposure and has limited availability. As such, a laboratory biomarker is desirable. This review briefly discusses the pathophysiology and management of bile acid diarrhoea and critically evaluates methods for its diagnosis, including serum 7α-hydroxy-4-cholesten-3-one, faecal bile acid measurement, serum fibroblast growth factor 19, urine-2-propanol, and the 14C-glycocholate breath and stool test.

A large deletion on CFA28 omitting ACSL5 gene is associated with intestinal lipid malabsorption in the Australian Kelpie dog breed.

Inborn errors of metabolism are genetic conditions that can disrupt intermediary metabolic pathways and cause defective absorption and metabolism of dietary nutrients. In an Australian Kelpie breeding population, 17 puppies presented with intestinal lipid malabsorption. Juvenile dogs exhibited stunted postnatal growth, steatorrhea, abdominal distension and a wiry coat. Using genome-wide association analysis, an associated locus on CFA28 (Praw = 2.87E-06) was discovered and validated in a closely related population (Praw = 1.75E-45). A 103.3 kb deletion NC_006610.3CFA28:g.23380074_23483377del, containing genes Acyl-CoA Synthetase Long Chain Family Member 5 (ACSL5) and Zinc Finger DHHC-Type Containing 6 (ZDHHC6), was characterised using whole transcriptomic data. Whole transcriptomic sequencing revealed no expression of ACSL5 and disrupted splicing of ZDHHC6 in jejunal tissue of affected Kelpies. The ACSL5 gene plays a key role in long chain fatty acid absorption, a phenotype similar to that of our affected Kelpies has been observed in a knockout mouse model. A PCR-based diagnostic test was developed and confirmed fully penetrant autosomal recessive mode of inheritance. We conclude the structural variant causing a deletion of the ACSL5 gene is the most likely cause for intestinal lipid malabsorption in the Australian Kelpie.

A proton-coupled folate transporter mutation causing hereditary folate malabsorption locks the protein in an inward-open conformation.

The proton-coupled folate transporter (PCFT, SLC46A1) is required for folate intestinal absorption and transport across the choroid plexus. Recent work has identified a F392V mutation causing hereditary folate malabsorption. However, the residue properties responsible for this loss of function remains unknown. Using site-directed mutagenesis, we observed complete loss of function with charged (Lys, Asp, and Glu) and polar (Thr, Ser, and Gln) Phe-392 substitutions and minimal function with some neutral substitutions; however, F392M retained full function. Using the substituted-cysteine accessibility method (with N-biotinyl aminoethyl methanethiosulfonate labeling), Phe-392 mutations causing loss of function, although preserving membrane expression and trafficking, also resulted in loss of accessibility of the substituted cysteine in P314C-PCFT located within the aqueous translocation pathway. F392V function and accessibility of the P314C cysteine were restored by insertion of a G305L (suppressor) mutation. A S196L mutation localized in proximity to Gly-305 by homology modeling was inactive. However, when inserted into the inactive F392V scaffold, function was restored (mutually compensatory mutations), as was accessibility of the P314C cysteine residue. Reduced function, documented with F392H PCFT, was due to a 15-fold decrease in methotrexate influx Vmax, accompanied by a decreased influx Kt (4.5-fold) and Ki (3-fold). The data indicate that Phe-392 is required for rapid oscillation of the carrier among its conformational states and suggest that this is achieved by dampening affinity of the protein for its folate substrates. F392V and other inactivating Phe-392 PCFT mutations lock the protein in its inward-open conformation. Reach (length) and hydrophobicity of Phe-392 appear to be features required for full activity.

The Endosomal Recycling Pathway-At the Crossroads of the Cell.

The endosomal recycling pathway lies at the heart of the membrane trafficking machinery in the cell. It plays a central role in determining the composition of the plasma membrane and is thus critical for normal cellular homeostasis. However, defective endosomal recycling has been linked to a wide range of diseases, including cancer and some of the most common neurological disorders. It is also frequently subverted by many diverse human pathogens in order to successfully infect cells. Despite its importance, endosomal recycling remains relatively understudied in comparison to the endocytic and secretory transport pathways. A greater understanding of the molecular mechanisms that support transport through the endosomal recycling pathway will provide deeper insights into the pathophysiology of disease and will likely identify new approaches for their detection and treatment. This review will provide an overview of the normal physiological role of the endosomal recycling pathway, describe the consequences when it malfunctions, and discuss potential strategies for modulating its activity.

Enteric anendocrinosis attributable to a novel Neurogenin-3 variant.

Congenital diarrhea and enteropathies (CODEs) are a group of monogenic disorders that often present with severe diarrhea in the first weeks of life. Enteric anendocrinosis (EA), an extremely rare cause of CODE, is characterized by a marked reduction of intestinal enteroendocrine cells (EC). EA is associated with recessively inherited variants in Neurogenin-3 (NEUROG3) gene. Here we investigate a case of a male infant who presented with mysterious severe malabsorptive diarrhea since birth. Thorough clinical assessments and laboratory tests were successful to exclude the majority of differential diagnosis categories. However, the patient's diagnosis was not established until the genetic test using whole-exome sequencing (WES) was performed. We identified a novel homozygous missense disease-causing variant (DCV) in NEUROG3 (c.413C>G, p.Thr138Arg). Moreover, molecular dynamic simulation analysis showed that (p.Thr138Arg) led to a global change of the NEUROG3 orientation affecting its DNA binding capacity. To the best of our knowledge, this is the first time to apply WES to reach a differential diagnosis of patients with CODEs. Our study not only expands our knowledge about NEUROG3 variants and their clinical consequences but also proves that WES is a very effective tool for the diagnosis of CODEs. This might be of value in early diagnosis of diseases and prenatal CODEs detection.

Lysophosphatidic Acid Increases Maturation of Brush Borders and SGLT1 Activity in MYO5B-deficient Mice, a Model of Microvillus Inclusion Disease.

BACKGROUND & AIM: Myosin VB (MYO5B) is an essential trafficking protein for membrane recycling in gastrointestinal epithelial cells. The inactivating mutations of MYO5B cause the congenital diarrheal disease, microvillus inclusion disease (MVID). MYO5B deficiency in mice causes mislocalization of SGLT1 and NHE3, but retained apical function of CFTR, resulting in malabsorption and secretory diarrhea. Activation of lysophosphatidic acid (LPA) receptors can improve diarrhea, but the effect of LPA on MVID symptoms is unclear. We investigated whether LPA administration can reduce the epithelial deficits in MYO5B-knockout mice. METHODS: Studies were conducted with tamoxifen-induced, intestine-specific knockout of MYO5B (VilCreERT2;Myo5bflox/flox) and littermate controls. Mice were given LPA, an LPAR2 agonist (GRI977143), or vehicle for 4 days after a single injection of tamoxifen. Apical SGLT1 and CFTR activities were measured in Üssing chambers. Intestinal tissues were collected, and localization of membrane transporters was evaluated by immunofluorescence analysis in tissue sections and enteroids. RNA sequencing and enrichment analysis were performed with isolated jejunal epithelial cells. RESULTS: Daily administration of LPA reduced villus blunting, frequency of multivesicular bodies, and levels of cathepsins in intestinal tissues of MYO5B-knockout mice compared with vehicle administration. LPA partially restored the brush border height and the localization of SGLT1 and NHE3 in small intestine of MYO5B-knockout mice and enteroids. The SGLT1-dependent short-circuit current was increased and abnormal CFTR activities were decreased in jejunum from MYO5B-knockout mice given LPA compared with vehicle. CONCLUSIONS: LPA may regulate a MYO5B-independent trafficking mechanism and brush border maturation, and therefore be developed for treatment of MVID.

Gremlin 1 depletion in vivo causes severe enteropathy and bone marrow failure.

The intestinal epithelium is perpetually renewed from a stem cell niche in the base of crypts to maintain a healthy bowel mucosa. Exit from this niche and maturation of epithelial cells requires tightly controlled gradients in BMP signalling, progressing from low BMP signalling at the crypt base to high signalling at the luminal surface. The BMP antagonist gremlin 1 (Grem1) is highly expressed by subepithelial myofibroblasts adjacent to the intestinal crypts but its role in regulating the stem cell niche and epithelial renewal in vivo has not been explored. To explore the effects of Grem1 loss in adulthood following normal growth and development, we bred mice (ROSA26CreER-Grem1 flx/flx ) in which Grem1 could be deleted by tamoxifen administration. While Grem1 remained intact, these mice were healthy, grew normally, and reproduced successfully. Following Grem1 depletion, the mice became unwell and were euthanised (at 7-13 days). Post-mortem examination revealed extensive mucosal abnormalities throughout the small and large intestines with failure of epithelial cell replication and maturation, villous atrophy, and features of malabsorption. Bone marrow hypoplasia was also observed with associated early haematopoietic failure. These results demonstrate an essential homeostatic role for gremlin 1 in maintaining normal bowel epithelial function in adulthood, suggesting that abnormalities in gremlin 1 expression can contribute to enteropathies. We also identified a previously unsuspected requirement for gremlin 1 in normal haematopoiesis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

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

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.

Fermentable fibers induce rapid macro- and micronutrient depletion in Toll-like receptor 5-deficient mice.

Functional fermentable fibers are considered essential for a healthy diet. Recently, we demonstrated that gut microbiota dysbiotic mice fed an inulin-containing diet (ICD) developed hepatocellular carcinoma (HCC) within 6 mo. In particular, a subset of Toll-like receptor 5-deficient (T5KO) mice prone to HCC exhibited rapid onset of hyperbilirubinemia (HB) and cholemia; these symptoms provide rationale that ICD induces cholestasis. Our objective in the present study was to determine whether inulin-fed T5KO-HB mice exhibit other known consequences of cholestasis, including essential fatty acid and fat-soluble vitamin deficiencies. Here, we measured hepatic fatty acids and serum vitamin A and D levels from wild-type (WT), T5KO low bilirubin (LB) and T5KO-HB mice fed ICD for 4 wk. Additionally, hepatic RNAseq and proteomics were performed to ascertain other metabolic alterations. Compared with WT and T5KO-LB, T5KO-HB mice exhibited steatorrhea, i.e., ~50% increase in fecal lipids. This could contribute to the significant reduction of linoleate in hepatic neutral lipids in T5KO-HB mice. Additionally, serum vitamins A and D were ~50% reduced in T5KO-HB mice, which was associated with metabolic compromises. Overall, our study highlights that fermentable fiber-induced cholestasis is further characterized by depletion of macro-and micronutrients.NEW & NOTEWORTHY Feeding a dietary, fermentable fiber diet to a subset of Toll-like receptor 5 deficient (T5KO) mice induces early onset hyperbilirubinemia and cholemia that later manifests to hepatocellular carcinoma (HCC). Our study highlights that fermentable fiber-induced cholestasis is characterized with modest macro- and micronutrient deficiencies that may further contribute to hepatic biliary disease. Compared with chemical induction, immunization, surgery, or genetic manipulation, these findings provide a novel approach to study the cholestatic subtype of HCC.

Imerslund-Gräsbeck Syndrome presenting with microangiopathic hemolytic anemia in a child.

Imerslund-Gräsbeck Syndrome is a rare autosomal recessive disorder characterized by proteinuria and selective malabsorption of cobalamin. Deficiency of cobalamin can lead to megaloblastic anemia, pancytopenia and even "pseudo"-thrombotic microangiopathy (TMA). Signs of mechanical hemolysis on peripheral blood smear, elevated lactate dehydrogenase and thrombocytopenia are common findings of TMA. We report a child presenting with TMA features with cobalamin deficiency. Because of her family history of vitamin B12 deficiency and proteinuria, the performed genetic analysis revealed that an Imerslund-Gräsbeck Syndrome with the detection of a homozygous mutation in AMN gene.