Microvillus Inclusion Disease Research Articles

Microvillous Inclusion Disease—An Ultrastructural Diagnosis: With a Review of the Literature

Microvillous inclusion disease is a rare disorder of infancy associated with protracted diarrhea. This malady reveals distinct ultrastructural changes. The surface enterocytes of the duodenum show vesicles lined with microvilli and the surface microvilli are poorly formed. The authors present one case of microvillous inclusion disease with a review of the literature.

CD10 immunohistochemistry stains enteric mucosa, but negative staining is unreliable in the setting of active enteritis

Ileal pouch–anal anastomosis is the definitive therapy for ulcerative colitis that is refractory to medical treatment or that has developed neoplasia. Patients with this procedure are routinely followed using directed endoscopic biopsies to monitor for dysplasia in the rectal cuff, residual/recurrent ulcerative colitis, and nonspecific acute inflammation of the ileal pouch (pouchitis), which have different clinical management and outcomes. Thus, accurate localization of mucosal biopsies is crucial to a definitive histological diagnosis, but is complicated by overlapping clinical presentations of pouchitis and ulcerative colitis, post-surgical and inflammatory changes to the mucosa, and altered endoscopic anatomy, resulting in difficulty determining whether a mucosal biopsy is ileal or rectal in origin for both the endoscopist and the pathologist. We explored the utility of CD10 immunohistochemistry to aid diagnosis in this clinical setting by highlighting the enteric mucosa, based on previous studies showing its utility in brush border staining and in the diagnosis of microvillous inclusion disease. We found uniformly positive CD10 immunostaining in normal enteric mucosa, but variable loss of expression in the setting of active enteritis. Specifically, CD10 staining was lost in up to 10% of the mucosa in 1/12 ileostomies and 4/13 enteric anastomoses, in 10–80% of the mucosa in 9/10 cases of Crohn's ileitis, in 10–60% of the mucosa in 7/16 ileal pouches, and in 20–90% of the mucosa in 6/8 cases of backwash ileitis, usually in the presence of active inflammation. There was no CD10 expression by normal or diseased colonic mucosa. Therefore, while CD10 immunostaining identifies normal enteric mucosa with 100% specificity, negative staining does not definitively exclude small intestinal mucosa in the setting of active enteritis, a common condition in ileal pouch mucosa.

Microvillus Inclusion Disease: Prenatal Ultrasound Findings, Molecular Diagnosis and Genetic Counseling of Congenital Diarrhea

To present prenatal ultrasound findings and molecular diagnosis of microvillus inclusion disease, and to review the literature of abnormal prenatal ultrasound findings associated with congenital diarrhea. A 21-year-old woman, gravida 1, para 0, had generalized bowel dilation of the fetus on prenatal ultrasound at 29 gestational weeks. She and her husband were non-consanguineous, and there was no family history of congenital diarrhea. Prenatal ultrasound at 29 gestational weeks revealed a honeycomb appearance of the bowel without ascites or intraperitoneal calcification. At 36 gestational weeks, polyhydramnios dilated bowel loops were observed, and a 3,355-g male baby was delivered with a distended abdomen. Postnatally, the neonate suffered from watery diarrhea and abdominal distension but there was no mechanical bowel obstruction. An endoscopic biopsy of the small bowel revealed intracytoplasmic inclusions lined by intact microvilli in the apical surface of the intestinal epithelial cells consistent with the diagnosis of microvillus inclusion disease. Mutation analysis of blood samples of the neonate and parents revealed a heterozygous nonsense mutation of c.445C <T, p.Q149X in exon 4 of the MYO5B gene in the father and proband, and a heterozygous nonsense mutation of c.1021C < T, p.Q341X in exon 9 of the MYO5B gene in the mother and proband. Prenatal sonographic identification of dilated bowel loops in association with polyhydramnios suggests congenital diarrhea and a differential diagnosis of microvillus inclusion disease in addition to congenital chloride diarrhea and congenital sodium diarrhea. Molecular analysis of the MYO5B gene is helpful in genetic counseling and prenatal diagnosis of recurrent microvillus inclusion disease in subsequent pregnancies.

Microvillous Inclusion Disease: A Clinicopathologic Study of 17 Cases from the UK

Background: Microvillous inclusion disease (MVID) is a rare congenital disease producing intractable secretory diarrhea in early infancy. It is characterized by diffuse intestinal villous atrophy with no inflammatory reaction. Ultrastructural identification of apical microvillous inclusions in the surface enterocytes is diagnostic. However, there is difficulty in the diagnosis of MVID due to the existence of variants (e.g., microvillous dystrophy), possible disease resolution, and tissue orientation for electron microscopy (EM). The authors analyzed materials from 4 patients with MVID from a single institution. The morphologic features, distribution of lesions, biomarkers, and complementary ultrastructural characteristics were studied. Design: Materials of MVID cases were collected from 6 different hospitals in the United Kingdom between 1990 and 2008. Epidemiological data, including age range, median, mode, sex ratios, and follow-up, were retrieved. All intestinal biopsy specimens were analyzed histologically, histochemically (for PAS, n = 17), immunohistochemically (for CD10, n = 2 and polyclonal CEA, n = 4), and ultrastructurally (n = 9). Results: Ultrastructurally, apical microvillous inclusions in surface enterocytes in duodenal biopsies were identified in all cases, while 1 case had variant morphology (microvillous dystrophy and very occasionally atypical microvillous inclusions). Tissue orientation for EM was supportive for identification of inclusions in apical enterocytes. Morphologically, a bubbly vacuolated appearance of the apical cytoplasm with extensive or patchy absence of the brush border with occasional cytoplasm inclusions was observed in the enterocytes. Some of these changes vaguely resembled gastric mucin cell metaplasia. Architecturally, villous blunting with either crypt hypoplasia or hyperplasia and absence of inflammation were common findings. The epithelial changes were also found in colon biopsies. PAS, CD10, and p-CEA showed a bright apical cytoplasmic blush/staining, which correlated ultrastructurally with apical granules with inclusions of variable electron density in all cases. These stains also highlighted the targetoid inclusions. Conclusion: Besides electron microscopy identification of inclusions, the light microscopic morphological features together with the biomarker studies highlighting the apical cytoplasmic blush are quite unique and diagnostic of MVID. Furthermore, it is the opinion of the authors that a diagnosis of MVID can be made without electron microscopy.

Light Microscopic Diagnosis of Microvillus Inclusion Disease on Colorectal Specimens Using CD10

Microvillus inclusion disease (MID) is a rare neonatal enteropathy that is typically diagnosed using electron microscopy to show characteristic inclusions in conjunction with light microscopy and periodic acid-Schiff staining to show lack of the normal brush border on biopsies obtained endoscopically from the small bowel. MID has also been diagnosed using CD10 immunoreactivity that shows abnormal intense cytoplasmic staining in absorptive small bowel enterocytes. As ultrastructural studies also show abnormal microvillus inclusions in absorptive colonocytes in these patients, we investigated the use of CD10 immunoreactivity on colon specimens. We studied the CD10 staining pattern in 4 colon specimens from patients with MID and in colon biopsy specimens from pediatric control patients with and without histopathologic abnormalities. All MID cases had cytoplasmic CD10 staining in absorptive colonocytes in contrast to the control patients who did not show any epithelial CD10 staining. We conclude that abnormal cytoplasmic CD10 staining of absorptive colonocytes can aid in the diagnosis of MID, which may be invaluable in the situations where only colon biopsy specimens are available for examination.

M1242 Negative Capsule Endoscopy on Management of the Patients With Obscure Gastrointestinal Bleeding is Strongly Related to Anti-Platelet Agents Therapy

Congenital diarrheal diseases can be difficult to diagnose. Some diseases, such as microvillus inclusion disease and congenital tufting enteropathy (CTE), require biopsy diagnosis. CTE is characterized by intestinal epithelial cell dysplasia leading to severe malabsorption and significant morbidity1. The pathology in CTE is not uniform making diagnosis even more difficult. Last year, we identified mutations in the gene for epithelial cell adhesion molecule (EpCAM) in CTE2. Immunohistochemical staining for EpCAM was found to be absent in duodenal specimens from patients with CTE as compared with normal tissue. In this study we hypothesize that lack of EpCAM staining in the intestinal epithelium is specific to CTE. After IRB consent, fluorescent immunohistochemistry of available formaldehyde-fixed, paraffin-embedded duodenal biopsy tissue was performed. Tissue from 4 patients with Ulcerative Colitis, 4 patients with Crohn's disease, 4 patients with Celiac disease, 1 patient with microvillus inclusion disease, 2 patients with non specific duodenitis and 4 normal age matched controls was included. Immunohistochemical staining for EpCAM was performed using mouse monoclonal anti-EpCAM antibody (clone 323/A3; Abcam, Cambridge, MA). Fluorescent secondary antibody (mouse IgG) was applied. Isotype controls were also performed. EpCAM was found to be present in the epithelia of all tissue specimens except for those from patients with CTE and isotype controls. In this study we describe lack of EpCAM staining in patients with CTE that is not seen in other gastrointestinal diarrheal diseases. Immunohistochemical staining for EpCAM may be a useful tool in the evaluation of congenital and chronic diarrheal diseases and specifically in making the diagnosis of CTE. 1. Sherman, P.M., Mitchell, D.J. & Cutz, E. Neonatal enteropathies: defining the causes of protracted diarrhea of infancy. J Pediatr Gastroenterol Nutr 38, 16-26 (2004). 2. Sivagnanam M., Mueller JL, Lee H, Chen Z, Nelson SF, Turner D, Zlotkin SH, Pencharz PB, Ngan B., Libiger O.,Schork NJ, Lavine, JE, Taylor S., Newbury RO, Kolodner RD, Hoffman, HM Identification of EpCAM as the gene for congenital tufting enteropathy. Gastroenterology. 2008 Aug;135(2):429-37

Toward a cellular model of microvillus inclusion disease

Toward a cellular model of microvillus inclusion disease

Loss-of-function of MYO5B is the main cause of microvillus inclusion disease: 15 novel mutations and a CaCo-2 RNAi cell model

Autosomal recessive microvillus inclusion disease (MVID) is characterized by an intractable diarrhea starting within the first few weeks of life. The hallmarks of MVID are a lack of microvilli on the surface of villous enterocytes, occurrence of intracellular vacuoles lined by microvilli (microvillus inclusions), and the cytoplasmic accumulation of periodic acid-Schiff (PAS)-positive vesicles in enterocytes. Recently, we identified mutations in MYO5B, encoding the unconventional type Vb myosin motor protein, in a first cohort of nine MVID patients. In this study, we identified 15 novel nonsense and missense mutations in MYO5B in 11 unrelated MVID patients. Fluorescence microscopy, Western blotting, and electron microscopy were applied to analyze the effects of MYO5B siRNA knock-down in polarized, brush border possessing CaCo-2 cells. Loss of surface microvilli, increased formation of microvillus inclusions, and subapical enrichment of PAS-positive endomembrane compartments were induced in polarized, filter-grown CaCo-2 cells, following MYO5B knock-down. Our data indicate that MYO5B mutations are a major cause of microvillus inclusion disease and that MYO5B knock-down recapitulates most of the cellular phenotype in vitro, thus independently showing loss of MYO5B function as the cause of microvillus inclusion disease.

Ultrastructural Aspects of Enterocyte Defects in Infancy and Childhood

Although there has been substantial progress in the identification of diarrheal diseases in infancy and childhood, electron microscopy may be still required for establishing diagnosis, staging, and response to therapy. This review describes severe conditions in which histopathologic examination alone cannot provide a firm diagnosis needed for therapeutic decisions. Microvillus inclusion disease, in its several variants, typifies this category. In certain forms of congenital disorders of glycosylation with gastrointestinal involvement, electron microscopic diagnosis is helpful. Among disorders due to abnormal immune-mediated reactions, celiac disease and cow's milk protein intolerance show fine structural changes of both diagnostic and staging value. Likewise, protein-losing enteropathies, including lymphangectasia, reveal information on the nature and extent of intestinal involvement.

Current perspectives on pediatric intestinal transplantation

Irreversible intestinal failure in children is predominantly caused by surgical conditions such as volvulus, necrotizing enterocolitis, and gastroschisis. Functional intestinal failure from motility disorders such as intestinal pseudo-obstruction or enterocyte dysfunction with microvillus inclusion disease also may require intestine replacement. Approved indications for intestinal transplantation include liver dysfunction, loss of major venous access, frequent central line-related sepsis, and recurrent episodes of severe dehydration despite intravenous fluid management. Surgical options include transplantation of the isolated intestine, combined liver-intestine transplantation, or multivisceral transplantation of the stomach, duodenum, pancreas, and small bowel (with or without the liver). Immunosuppression for intestinal transplantation is based on tacrolimus therapy, often with induction immunosuppression using antilymphocyte antibodies (eg, antithymocyte antibody and alemtuzumab). Experience at centers of excellence demonstrates 1- and 5-year patient survival rates of 95% and 77%, respectively, with ongoing investigations focusing on lowering long-term causes of graft loss such as chronic rejection.

393 Apical Recycling Endosome-Associated Myosin Vb Is Mutated in Microvillus Inclusion Disease and Is Involved in Intestinal Brush Border Development

Background & Aims: Microvillus inclusion disease is a rare and fatal congenital enteropathy, presenting with intractable secretory diarrhea shortly after birth. The complete inability to absorb nutrients from intestinal lumen demands total parenteral nutrition, and, eventually, transplantation of the small intestine. MID characteristics varies among patients and generally comprises of villous atrophy and crypt hyperplasia, and, at the cellular level, by the apical brush border atrophy, accumulation of apical proteins, lysosomes and microvilli-like inclusions in the apical cytoplasm of intestinal absorptive cells. Previously we have shown that MID enterocytes display abnormal expression of apical recycling endosomal markers, i.e. Rab11a, FIP-1 (RCP), FIP-5 (Rip11), resulting in a defective apical recycling system in MID. In this study, we aimed to identify the genetic cause and functional consequences that underlie the microvillus inclusion disease. Methods: We screened the genomic DNA of three patients diagnosed with microvillus inclusion disease, their siblings and parents. Biopsies of small intestine from MID and control patients were used to analyze the organization of organelles and localization of proteins involved in intracellular trafficking of brush border proteins. Results: In all MID patients together we have identified two substitutions, one deletion, and two protein truncating mutations in the myosin 5B gene. The MYO5B encodes for an actin filament-binding molecular motor protein that interacts with the small GTPase Rab11a, a marker of recycling endosomes, and thus facilitates the intracellular trafficking of apical proteins towards the apical membrane. We also found aberrant expression and subcellular distribution of myosin Vb protein and other key proteins that interact withmyosin Vb and/or control apical recycling endosome-mediated protein trafficking. Conclusions: The endosomal system that ensures the recycling of brush border proteins, with myosin Vb as a critical regulator, is required to develop and maintain functional apical cell surface in human enterocytes, and perturbations in this can be causally linked to microvillus inclusion disease. Mutations occurring at different positions of MYO5B gene and thus affecting different functional regions of MYO5B protein could explain the diversity of phenotypes present in MID patients. The identification of mutations in MYO5B as the cause for MID brings a major advance in setting the reliable diagnosis, enables the genetic counseling and prenatal screening, as well as paves the way for developing alternative therapeutic strategies.

394 Intestine Specific Deletion of N-WASP Leads to Alteration of Gut Homeostasis in Mice

Background & Aims: Microvillus inclusion disease is a rare and fatal congenital enteropathy, presenting with intractable secretory diarrhea shortly after birth. The complete inability to absorb nutrients from intestinal lumen demands total parenteral nutrition, and, eventually, transplantation of the small intestine. MID characteristics varies among patients and generally comprises of villous atrophy and crypt hyperplasia, and, at the cellular level, by the apical brush border atrophy, accumulation of apical proteins, lysosomes and microvilli-like inclusions in the apical cytoplasm of intestinal absorptive cells. Previously we have shown that MID enterocytes display abnormal expression of apical recycling endosomal markers, i.e. Rab11a, FIP-1 (RCP), FIP-5 (Rip11), resulting in a defective apical recycling system in MID. In this study, we aimed to identify the genetic cause and functional consequences that underlie the microvillus inclusion disease. Methods: We screened the genomic DNA of three patients diagnosed with microvillus inclusion disease, their siblings and parents. Biopsies of small intestine from MID and control patients were used to analyze the organization of organelles and localization of proteins involved in intracellular trafficking of brush border proteins. Results: In all MID patients together we have identified two substitutions, one deletion, and two protein truncating mutations in the myosin 5B gene. The MYO5B encodes for an actin filament-binding molecular motor protein that interacts with the small GTPase Rab11a, a marker of recycling endosomes, and thus facilitates the intracellular trafficking of apical proteins towards the apical membrane. We also found aberrant expression and subcellular distribution of myosin Vb protein and other key proteins that interact withmyosin Vb and/or control apical recycling endosome-mediated protein trafficking. Conclusions: The endosomal system that ensures the recycling of brush border proteins, with myosin Vb as a critical regulator, is required to develop and maintain functional apical cell surface in human enterocytes, and perturbations in this can be causally linked to microvillus inclusion disease. Mutations occurring at different positions of MYO5B gene and thus affecting different functional regions of MYO5B protein could explain the diversity of phenotypes present in MID patients. The identification of mutations in MYO5B as the cause for MID brings a major advance in setting the reliable diagnosis, enables the genetic counseling and prenatal screening, as well as paves the way for developing alternative therapeutic strategies.

396 The ZO-1 Actin Binding Region (ABR) Is Required for Cytoskeletal Tight Junction Regulation

Background & Aims: Microvillus inclusion disease is a rare and fatal congenital enteropathy, presenting with intractable secretory diarrhea shortly after birth. The complete inability to absorb nutrients from intestinal lumen demands total parenteral nutrition, and, eventually, transplantation of the small intestine. MID characteristics varies among patients and generally comprises of villous atrophy and crypt hyperplasia, and, at the cellular level, by the apical brush border atrophy, accumulation of apical proteins, lysosomes and microvilli-like inclusions in the apical cytoplasm of intestinal absorptive cells. Previously we have shown that MID enterocytes display abnormal expression of apical recycling endosomal markers, i.e. Rab11a, FIP-1 (RCP), FIP-5 (Rip11), resulting in a defective apical recycling system in MID. In this study, we aimed to identify the genetic cause and functional consequences that underlie the microvillus inclusion disease. Methods: We screened the genomic DNA of three patients diagnosed with microvillus inclusion disease, their siblings and parents. Biopsies of small intestine from MID and control patients were used to analyze the organization of organelles and localization of proteins involved in intracellular trafficking of brush border proteins. Results: In all MID patients together we have identified two substitutions, one deletion, and two protein truncating mutations in the myosin 5B gene. The MYO5B encodes for an actin filament-binding molecular motor protein that interacts with the small GTPase Rab11a, a marker of recycling endosomes, and thus facilitates the intracellular trafficking of apical proteins towards the apical membrane. We also found aberrant expression and subcellular distribution of myosin Vb protein and other key proteins that interact withmyosin Vb and/or control apical recycling endosome-mediated protein trafficking. Conclusions: The endosomal system that ensures the recycling of brush border proteins, with myosin Vb as a critical regulator, is required to develop and maintain functional apical cell surface in human enterocytes, and perturbations in this can be causally linked to microvillus inclusion disease. Mutations occurring at different positions of MYO5B gene and thus affecting different functional regions of MYO5B protein could explain the diversity of phenotypes present in MID patients. The identification of mutations in MYO5B as the cause for MID brings a major advance in setting the reliable diagnosis, enables the genetic counseling and prenatal screening, as well as paves the way for developing alternative therapeutic strategies.

Index of Suspicion

Index of Suspicion

Navajo microvillous inclusion disease is due to a mutation inMYO5B

Microvillous Inclusion Disease (MID) is a rare, autosomal recessive gastrointestinal disease of increased frequency among the Navajos. Previous work has shown a deficiency of RAB8 in one Japanese patient, while homozygous mutations in MYO5B were found in 7 of 10 mostly Middle Eastern families. We have identified a shared homozygous mutation in MYO5B in seven affected Navajos with the expected heterozygosity in five parents. We have developed a simple restriction enzyme based assay that allows for rapid screening for this mutation.

MYO5B mutations cause microvillus inclusion disease and disrupt epithelial cell polarity

Following homozygosity mapping in a single kindred, we identified nonsense and missense mutations in MYO5B, encoding type Vb myosin motor protein, in individuals with microvillus inclusion disease (MVID). MVID is characterized by lack of microvilli on the surface of enterocytes and occurrence of intracellular vacuolar structures containing microvilli. In addition, mislocalization of transferrin receptor in MVID enterocytes suggests that MYO5B deficiency causes defective trafficking of apical and basolateral proteins in MVID.

Novel NaPi-2c mutations that cause mistargeting of NaPi-2c protein and uncoupling of Na-Picotransport cause HHRH

hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is an autosomal recessive inherited disorder of mineral and bone metabolism. It is characterized by hypophosphatemia, rickets, and increased serum 1,25-dihydroxyvitamin D concentration, resulting in secondary absortive hypercalciuria and

Microvillus Inclusion Disease Associated With Coarctation of the Aorta and Bicuspid Aortic Valve

Microvillus inclusion disease is a life-threatening diarrheal disorder of infancy characterized by the presence of microvillus inclusions within the intestinal epithelium. We report a case of a neonate with microvillus inclusion disease that was associated with coarctation of the aorta and bicuspid aortic valve, cardiac malformations within the spectrum of left ventricular outlet tract obstruction. Possible links between the intestinal and cardiac phenotypes are discussed.

599 Abnormal Expression of Apical Recycling Endosome Proteins in Microvillous Inclusion Disease

599 Abnormal Expression of Apical Recycling Endosome Proteins in Microvillous Inclusion Disease

Caught in the traffic

In mice, deletion of the Rab8 protein disrupts organized molecular distribution to membranes of intestinal epithelial cells. Death by starvation follows, exactly as it does in humans with microvillus inclusion disease.