Recent advances in molecular pathology of Microvillus inclusion disease (MVID)

Cornelia Thoeni, MD,PhD Division of Gastroenterology, Hepatology and Nutrition, Hospital for Sick Children, Toronto, Ontario,Canada E-Mail: corneliathoeni@utanet.at Ernest Cutz, MD Division of Pathology, Department of Pediatric Laboratory Medicine, The Research Institute, The Hospital for Sick Children and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada E-Mail: ernest.cutz@sickkids.ca Definition

Congenital diarrheal disorders are a group of rare chronic enteropathies due to a variety of

etiologies. Based on specific molecular defects, over 30 entities have been identified to-date

(1).These conditions have been classified into four major groups based on the nature of the

cellular/molecular defect and include (a) defects of digestion, absorption and transport of

nutrients and electrolytes; (b) defects of enterocyte differentiation and polarization; (c) defects of

enteroendocrine cell differentation and (d) defects of modulation of intestinal immune response

(ie autoimmune enteropathy). Microvillus inclusion disease (MVID), prototypical of the defects

of enterocyte differentiation and polarity, was the first congenital enteropathy described in 1978

as a familial disorder characterized on transmission electron microscopy (TEM) by the presence

of peculiar intracytoplasmic inclusions in enterocytes (2).The term MVID was coined in 1989

when further cases were identified and their familial/genetic origin confirmed (3).

To-date approxiamtely 70 cases are reported with hot spots in familial ethnicity in the Middle

East, French and Navajo Indians in North America (4-5). However the incidence of MVID

could be higher since the disease may be sometimes missed due to non specific clinical

manifestations which makes the diagnosis challenging.

Clinical presentation

Patients with MVID usually present with severe malabsorption and metabolic acidosis, because

of intractable watery diarrhea starting a few days after birth (early-onset disease or classical

MVID) or within first months of life (late-onset disease) (6). Although most patients with

MVID present during infancy and survive only on chronic TPN or bowel transplant. There are

exceptional cases reported with improvement in symptoms and survival in early adulthood

(relapsing and remitting phenotype) (6-7). Since the clinical presentation of MVID is similar

to other congenital enteropthies detailed analysis by histology, immunohistochemistry and

electron microscopy is essential finally to arrive at a correct diagnosis.

Histopathology

On routine HE staining, duodenal biopsy samples from patients with classical MVID show

partial to total villous atrophy and intracytoplasmic vacuoles in villous enterocytes (Figures 1A

&B).There is minimal or no inflammation in the lamina propria and there is lack of crypt

hyperplasia (ie normoplastic atrophy). In patients with variant form or with relapsing MVID,

duodenal biopsies may show partial villous atrophy or even normal villi. In these cases

cytoplasmic vacuolation of enerocytes may be patchy. The PAS stains, that in normal small

intestine demonstrates general acidic structures such as the apical brush border and the goblet

cells, in biopsies from MVID patients, this acidic material is mislocalized in the cytoplasm of

enterocytes, likely corresponding to secretory like granules, observed on EM (Figures 1 C&D).

Immunohistochemistry of duodenal biopsies from MVID patients shows cytoplasmic

mislocalization of the brush border proteins CD10, a neutral membrane-associated peptidase as

well as villin, a brush border microvillus associated intermediate filament (Figures 1 E&F).

Immunopositive, doughnut-like intracytoplasmic structures representing microvillous inclusions

are present in about 10% of villous enterocytes, but not in crypt cells. At light microscopy level,

such inclusions are best demonstrated by immunostaning for villin (Figure 2F) or by multilabel

immunoflorescence method combined with confocal microscopy (Figure 2G).

Electron microscopy (EM) examination of duodenal biopsies is essential for the confirmation of

diagnosis of MVID and for differentiation from other forms of congenital eneropathies.

Compared to normal EM features of duodenal enterocytes (Figures 2A&B), the typical EM

findings in MVID include reduced or absent brush border microvilli on apical membrane of

mature villous enterocytes (Figure 2C). Additional EM features include the presence of

microvillous inclusions (MI) with an internal lumen lined by normally appearing brush border

microvilli (Figures 2 C&D). MI are usually found in supranuclear cytoplasm, but partial

inclusions or aberrant microvilli can be also found on the basolateral or even basal plasma

membrane. Furtheremore there is increased number of variable autophagocytic-like inclusions

(vesicular bodies, Figure 2 C) and electron dense cytoplasmic granules (Figure E).

In contrast to villous enterocytes, crypt cells appear relatively normal except for increased

number of electron dense secretory-like granules. Due to sparse distribution of MI, diligent

search examining multiple blocks and deeper sections is often necessary, particularly in cases of

variant form of MVID. The spectrum of additional EM features seen in cases of MVID have

been reported (9).

Genetics

Mutations in myosin Vb (MYO5B) gene as a basis of MVID was first reported in 2008 (10).

MYO5B is a non-conventional myosin motor protein that facilitates protein trafficking and

recycling in polarized epithelial cells by Rab11-dependent mechanisms. Of the 41 published

mutations in the MYO5B gene identified in patients with MVID, 16 mutations were

homozygous, 9 mutations were heterozygous and additional 16 mutations were found in patients

with compond heterozygous mutations (10-11).

More recently, mutations in syntaxin 3 (STX3) gene were indentified as the disease causing

genes in 2 cases of variant form of MVID (12). These genes are coding proteins involved in

the regulation of intracellular traffic and exocytosis of vesicles in epithelial cells. STX3 is an

apical receptor protein involved in membrane fusion of enterocytes.

Cellular changes and defects in experimental models

In in vivo studies of biopsy samples from MVID patients harboring loss-of-function mutations in

the MYO5B or the STX3 gene, MVID was classified as a disorder of defective intracellular

traffic and disrupted cell polarity (10-13). Those cellular changes were validated by a

MYO5B RNAi as well as a STX3 RNAi CaCo2 cell model (10-13). Loss of a functional

myosinVb motor and syntaxin3 protein resulted in loss of apical microvilli mislocalized

microvilli in cytoplasmic inclusion bodies as well as secretory like granules (electron dense

structures in the cytoplasm). Furthermore those ultrastructural changes resulted from

redistribution of cell organelles as lysosomes, transporter proteins as well as small GTPases

regulating intracellular vesicle transport and establishment of a functional intestinal barrier (13).

These cellular alterations caused by genetic defects in the MYO5B or STX3 gene explain mainly

the clinical manifestation of chronic diarrhea due to a non-functional surface epithelium in the

small intestine (10-13).

The summary of cellular changes and resulting cell defects in both MYO5B and STX3 mutations

are shown in a schematic diagram (Figure 3).

It is hoped that with better understanding of basic cellular and molecular defects it will be

possible to develop pharmacologic treatment and or gene/stem cell therapies for MVID to

provide a viable alternative to chronic TPN or bowel transplantation.

References

1) Canani RB,Terrin G et al. Congenital diarrheal disorders:improved understanding of gene defects is leading to advances in intestinal physiology and clinical management. JPGN 2010 50:360-366. 2) Davidson GP, Cutz E et al. Familial enteropathy:a syndrome of protracted diarrhea from birth,failure to thrive and hypoplastic villous atrophy. Gastroenerology,1978,75:783-790. 3) Cutz E, Rhoads JM, et al. Microvillus inclusion disease: an inherited defect of brush-border assembly and differentiation. The New England Journal of Medicine 1989;320(10):646-651 4) Ruemmele FM, Schmitz J et al. Microvillous inclusion disease (microvillous atrophy). Orphanet J Rare Dis 2006, 1750-1172-1-22 5) Ericson R P, Larson-Thome K et al. Navajo microvillous inclusion disease is due to mutation in MYO5B. Am J Med Genetics, 2008 A 146 A: 3117-3119. 6) Sherman PM, Mitchell DJ et al. Neonatal enteropathies: defining the causes of protracted diarrhea of infancy. Journal of pediatric gastroenterology and nutrition 2004; 38(1):16-26 7) Croft MC, Howatson AG et al. Microvillous inclusion disease: an evolving condition. JPGN 2000,31:185-189. 8) Cutz E, Sherman PM. at al. Enteropathies associated with protracted diarrhea of infancy: clinicopathological features, cellular and molecular mechanisms. Pediatr Pathol Lab Med. 199717 (3): 335 – 68 9) Iancu TC, Mahajnah M et al. Microvillous inclusion disease: ultrastructural variability. Ultrastrzc Pathol. 2007 31 (3): 173-88 10) Muller T, Hess MW, et al. MYO5B mutations cause microvillus inclusion disease and disrupt epithelial cell polarity. Nature Genetics 2008; 40 (10):1163-1165. 11) Ruemmele FM, Muller T, et al. Loss-of-function of MYO5B is the main cause of microvillus inclusion disease: 15 novel mutations and a CaCo-2 RNAi cell model. Human mutation 2010; 31(5):544-551. 12) Wiegerinck CL, Janecke AR, et al. Loss of syntaxin 3 causes variant microvillus inclusion disease. Gastroenterology 2014 147 (1): 65-68. 13) Thoeni, C. E., G. F. Vogel et al. Microvillus inclusion disease: loss of myosin Vb disrupts intracellular traffic and cell polarity. Traffic 2014.15 (1):22-42. 14) Knowles BC, JR Goldenring et al. Apical Vesicle Trafficking Takes Center Stage in Neonatal Enteropathies. Gastroenterology, Editorials, Gastroenterology 2014;147:15–30

Figures A B

C D

E F G

Figure 1. Histopathology and immunohistochemistry of duodenal biopsy from classical case of MVID. (A) Hematoxylin and Eosin (HE) stain shows partial villous atrophy and (B) cytoplasmic vacuolation of enterocytes (black arrow). (C&D) PAS stain demonstrates diffuse positive staining of apical cytoplasm of enterocytes, (D) close up of PAS positive granules

(white arrow) in the cytoplasm of duodenal enterocytes. Immunohistochemistry of brush border markers (E) CD10 and (F) villin show cytoplasmic CD10 and villin positive inclusions in the cytoplasm of enterocytes (black arrows). (G) Confocal microscopy shows immunofluorescence staining for actin cytoskeleton with phalloidin 488 (ALEXA 488, green) and for lysosomes with Lamp2 –lysosomal associated membrane protein 2 (ALEXA 568, red). Duodenal enterocytes show actin-positive inclusion representing a microvillus inclusion (green ring) and prominent lysosomes (white arrow) in the cytoplasm. A B

C D

E

Figure 3b: TEM analysis of the duodenum of the STX3 MVID patient. Electron dense cytoplasmic vesicular structures represent secretory like granules. Goblet cells are marked with a red cross. Scale bar = 2 µm.

Figure 2 (A&B): Transmission electron microscopy (TEM) of a duodenal biopsy from a healthy control showing normal dense apical brush border microvilli (MI). Scale bar = 2 µm.

C – E: TEM analysis of a duodenal biopsy from a patient with MVID C: Low magnification of duodenal enterocytes show a intracytoplasmic microvillous inclusion. MV (microvilli), VB (vesicular bodies), NU (nucleus) D: Higher magnification of duodenal enterocytes showing an intracytoplasmic microvillous inclusion surrounded by prominent lysosomes (white arrow) E: High magnification of duodenal enterocytes showing apical loss of microvilli (black arrows) and intracytoplasmic secretory-like granules (electron dense granules).

Why toddlers with “atypical” MVID have a milder phe-notype when compared with “typical”MVID patients has notbeen definitively determined, but it seems to be most likelyowing to the association of STX3 with Rab11a-positiverecycling vesicles. Because STX3 does not participate inRab8a-positive vesicle fusion, Rab8a-dependent apical traf-ficking may not be affected by mutations in STX3, and as

result only a partial MVID phenotype was observed in thesepatients with STX3 mutations. STX3 is localized specificallyto the apical surface of epithelial cells, which is similar to thelocalization of MYO5B, and this localization likely accountsfor the lack of initial extra-intestinal manifestation of thisdisease.17,18 However, the epithelial cells of the kidney andstomach are highly polarized, and depend on the fusion of

Figure 1. Loss of STX3 inhibits MYO5B-dependent Rab11a-positive vesicle fusion to the apical plasma membrane and causesatypical microvillus inclusion disease (MVID). (A) Reclassification of MVID into typical and atypical MVID based on diseaseonset, gene mutation, diarrhea type, feeding tolerance, and microvillus inclusion presentation. (B) Left, Normal enterocyte withproper vesicle trafficking of cargo. Right, Magnified image of ARE and its predicted interaction with macropinosomes. BothRab8a and Rab11a bind to MYO5B directly on the ARE. Mutations in MYO5B that cause MVID obstruct MYO5B motorfunction, and prevent all subsequent apical trafficking via MYO5B-dependent pathways. Rab8a facilitates MYO5B-dependentapical trafficking and recycling, and has also been shown to activate Cdc42 via Tuba, which regulates cellular polarity, andfacilitates actin polymerization. Rab11a facilitates apical trafficking and recycling via MYO5B-dependent pathways. Rab11aalso normally regulates actin dynamics in a MYO5B-dependent manner, and is required to recycle microvilli after macro-pinocytosis. This recycling prevents microvillus inclusion formation after macropinocytosis of the microvilli. STX3 is requiredfor final Rab11a-positive vesicle fusion to the apical surface and mutations in STX3 disrupt the normal function of this pathway.All of these pathways together aid in microvilli growth and the maintenance of apical polarity.

EDITORIALS

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Figure 3: Schematic representation of cellular/molecular defects in MVID due to MYO5B and STX3 mutations (according to reference 14) Left, Normal enterocyte with proper vesicle trafficking of cargo. Right, Magnified image of ARE and its predicted interaction with macropinosomes. Both Rab8a and Rab11a bind to MYO5B directly on the ARE. Mutations in MYO5B that cause MVID obstruct MYO5B motor function and prevent all subsequent apical trafficking via MYO5B-dependent pathways. Rab8a facilitates MYO5B-dependent apical trafficking and recycling, and has also been shown to activate Cdc42 via Tuba, which regulates cellular polarity, and facilitates actin polymerization. Rab11a facilitates apical trafficking and recycling via MYO5B-dependent pathways. Rab11a also normally regulates actin dynamics in a MYO5B-dependent manner, and is required to recycle microvilli after macro- pinocytosis. This recycling prevents microvillus inclusion formation after macropinocytosis of the microvilli. STX3 is required for final Rab11a-positive vesicle fusion to the apical surface and mutations in STX3 disrupt the normal function of this pathway.All of these pathways together aid in microvilli growth and the maintenance of apical polarity.