Functional Characterization of Mutations in the Myosin Vb Gene Associated With Microvillus Inclusion Disease

Abstract

Objectives-Microvillus inclusion disease (MVID) is a rare autosomal recessive enteropathy characterized by intractable diarrhea and malabsorption. Recently, various MYO5B gene mutations have been identified in MVID patients. Interestingly, several MVID patients showed only a MYO5B mutation in one allele (heterozygous) or no mutations in the MYO5B gene, illustrating the need to further functionally characterize the cell biological effects of the MYO5B mutations.Methods-The genomic DNA of nine patients diagnosed with microvillus inclusion disease was screened for MYO5B mutations, and qPCR and immunohistochemistry on the material of two patients was performed to investigate resultant cellular consequences.Results-We demonstrate for the first time that MYO5B mutations can be correlated with altered myosin Vb mRNA expression and with an aberrant subcellular distribution of the myosin Vb protein. Moreover, we demonstrate that the typical and myosin Vb-controlled accumulation of rab11a-and FIP5-positive recycling endosomes in the apical cytoplasm of the cells is abolished in MVID enterocytes, which is indicative for altered myosin Vb function. Also, we report 8 novel MYO5B mutations in 9 MVID patients of various etnic backgrounds, including compound heterozygous mutations.Conclusions-Our functional analysis indicate that MYO5B mutations can be correlated with an aberrant subcellular distribution of the myosin Vb protein and apical recycling endosomes which,

Enfermedad por inclusión microvellositaria como causa de diarrea congénita severa. Caso clínico

“…En el año 2008 se identificó una mutación común en la mayoría de los pacientes investigados en el gen MYO5B que codifica para la proteína Myosin Vb, la cual se expresa en todos los epitelios y que es involucrada en el transporte de proteínas hacia la superficie celular y en la polaridad celular 11,12,13,14 . Se postula, por lo tanto, que el impedimento del transporte de proteí-nas claves será la causa de la enfermedad alterando así el correcto desarrollo y la regeneración de la membrana epitelial.…”

“…Con la microscopia electrónica se reconocen los cambios patognomónicos que son vacuolas intracitoplasmáticas con microvellosidades incluidas y ausencia o escasez de microvellosidades en el borde luminal. También se puede practicar una tinción inmunoreactiva que reconoce una peptidasa neutra CD-10 que en esta enfermedad se ubica dentro del citoplasma del enterocito en contraste con el intestino normal u otras patologías intestinales en las cuales suele demostrarse en la superficie 8,9,10 .En el año 2008 se identificó una mutación común en la mayoría de los pacientes investigados en el gen MYO5B que codifica para la proteína Myosin Vb, la cual se expresa en todos los epitelios y que es involucrada en el transporte de proteínas hacia la superficie celular y en la polaridad celular 11,12,13,14 . Se postula, por lo tanto, que el impedimento del transporte de proteí-nas claves será la causa de la enfermedad alterando así el correcto desarrollo y la regeneración de la membrana epitelial.…”

Myosin Vb and rab11a regulate ezrin phosphorylation in enterocytes

“…By contrast, other ezrin-phosphorylating kinases appear to, at least partially, localize to undefined intracellular compartments. For instance, following the activation of the polarity protein LKB1, Mst4 phosphorylates T567 in ezrin after being translocated to the enterocyte subapical domain by an unknown mechanism (ten Klooster et al, 2009). Also, the location of the active aPKCi is not clear while its activating kinase phosphoinositide-dependent kinase-1 (PDK1) associates with apical endosomes (Mashukova et al, 2012).…”

“…Proteins move up and down the microvilli membrane in response to signals and microvilli generate vesicles that are shed into the apical lumen (McConnell et al, 2009) where they regulate epithelial-microbial interactions (Shifrin et al, 2012). Interestingly, (part of) the molecular pathway(s) for the development of microvilli has been demonstrated to constitute a separate branch of the epithelial cell polarity program driven by the serine/threonine liver kinase B1 (LKB1) (Gloerich et al, 2012;ten Klooster et al, 2009). Whether the apically enriched Rab11a-positive recycling endosomes play a direct role in the development and organization of microvilli at the apical plasma membrane is not known.…”

“…Several kinases have been implicated in the direct T567 phosphorylation of ezrin in intestinal epithelial cells, including protein kinase B2/Akt2 (Shiue et al, 2005), atypical protein kinase C-iota (aPKCi) (Wald et al, 2008), mammalian Sterile 20 (Ste20)-like kinase-4 (Mst4) (Gloerich et al, 2012;ten Klooster et al, 2009), lymphocyte-oriented kinase (LOK) and Ste20-like kinase (SLK) (Viswanatha et al, 2012). Knockdown of each of these kinases in various intestinal epithelial cell models inhibits microvillus development at the apical plasma membrane (Gloerich et al, 2012;Shiue et al, 2005;ten Klooster et al, 2009;Viswanatha et al, 2012;Wald et al, 2008). Some of these kinases have been reported to localize to the apical microvillus membrane (Shiue et al, 2005;Viswanatha et al, 2012).…”