A Founder Mutation in <i>VPS11</i> Causes an Autosomal Recessive Leukoencephalopathy Linked to Autophagic Defects

<div><p>Genetic leukoencephalopathies (gLEs) are a group of heterogeneous disorders with white matter abnormalities affecting the central nervous system (CNS). The causative mutation in ~50% of gLEs is unknown. Using whole exome sequencing (WES), we identified homozygosity for a missense variant, <i>VPS11</i>: c.2536T>G (p.C846G), as the genetic cause of a leukoencephalopathy syndrome in five individuals from three unrelated Ashkenazi Jewish (AJ) families. All five patients exhibited highly concordant disease progression characterized by infantile onset leukoencephalopathy with brain white matter abnormalities, severe motor impairment, cortical blindness, intellectual disability, and seizures. The carrier frequency of the <i>VPS11</i>: c.2536T>G variant is 1:250 in the AJ population (n = 2,026). VPS11 protein is a core component of HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering) protein complexes involved in membrane trafficking and fusion of the lysosomes and endosomes. The cysteine 846 resides in an evolutionarily conserved cysteine-rich RING-H2 domain in carboxyl terminal regions of VPS11 proteins. Our data shows that the C846G mutation causes aberrant ubiquitination and accelerated turnover of VPS11 protein as well as compromised VPS11-VPS18 complex assembly, suggesting a loss of function in the mutant protein. Reduced <i>VPS11</i> expression leads to an impaired autophagic activity in human cells. Importantly, zebrafish harboring a <i>vps11</i> mutation with truncated RING-H2 domain demonstrated a significant reduction in CNS myelination following extensive neuronal death in the hindbrain and midbrain. Thus, our study reveals a defect in <i>VPS11</i> as the underlying etiology for an autosomal recessive leukoencephalopathy disorder associated with a dysfunctional autophagy-lysosome trafficking pathway.</p></div

The VPS C846G mutation decreases the stability of VPS11 protein.

<p>HeLa cells were transfected with 0.5 ug DNA of empty vector (Ctrl), FLAG-tagged VPS11-WT or VPS11-C846G. (A) Expression of transfected plasmids was examined at 48 hrs post-transfection by Western blot analysis. (B) The chase started 24 hrs post-transfection and was allowed for the indicated time. Blots are representatives of four independent experiments. IB: Immunoblot. (C) The percentage of protein decay was graphically reported and the difference of the <i>in cellulo</i> half-life between the WT and C846 mutant of VPS11 was analyzed in (D). The statistical significance was determined by an unpaired student <i>t</i> test. Results are the mean±SEM of four independent experiments. (E) Images were prepared with PyMOL (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>) using coordinates from the Protein Model Portal (VPS11_Q9H270, PDB 1iymA). (F) Far-UV CD spectra of the purified WT and C846G RING domain fragment proteins (amino acids 821–860). Results are the mean of three independent experiments. (G) HeLa cells were transfected with 2.0 ug DNA of empty vector, 1.0 ug of FLAG-tagged VPS11-WT with 1.0 ug of empty vector or 2.0 ug of FLAG-VPS11-C846G. Immunoprecipitations (IPs) were performed 48 hrs post-transfection and followed by Western blot analysis. Blots are representatives of four independent experiments. (H-I) The percentage of VPS11 ubiquitination was calculated and the difference of the <i>in cellulo</i> total ubiquitination profile was verified. The statistical significance was determined by an unpaired student <i>t</i> test. Results are the mean±SEM of four independent experiments. Data were considered significant when P values were <0.05 (*), <0.01(**) or <0.001 (***).</p

Significant cell death is observed in the CNS of zebrafish <i>vps11(plt)</i> mutants.

<p>TUNEL assay was performed on cryosectioned CNS tissue from <i>vps11(plt)</i> mutants and wild-type siblings at 3, 5, and 7 days post-fertilization (dpf). (A-C) Minimal apoptotic TUNEL+ cells were observed in the hindbrain of <i>vps11(plt)</i> mutants at 3, 5, and 7 dpf, respectively. (D-E) Tissue section of the midbrain of a wild-type control animal at 7 dpf showing minimal cell death (panel E, arrow). (F-G) Tissue section of the midbrain of a <i>vps11(plt)</i> mutant at 7 dpf showing extensive cell death. (H) Quantification of the average number of TUNEL+ cells observed in the hindbrain in <i>vps11(plt)</i> mutants and wild-type siblings at 3, 5, and 7 dpf. No significant differences were observed. (I) Quantification of the average number of TUNEL+ cells observed in the midbrain in <i>vps11(plt)</i> mutants and wild-type siblings at 3, 5, and 7 dpf. Asterisk indicates significantly different from control.</p

Major leukoencephalopathy manifestations in five individuals from three AJ families with homozygous <i>VPS11</i>: c.2536T>G (p.C846G).

<p>Major leukoencephalopathy manifestations in five individuals from three AJ families with homozygous <i>VPS11</i>: c.2536T>G (p.C846G).</p

Progressive loss of myelination in zebrafish <i>vps11(plt)</i> mutants.

<p>Cryosectioned CNS tissue from <i>vps11(plt)</i> mutants and wild-type siblings were immunolabeled with Mbp (myelin; green) and HuCD (neurons; red) at 3 and 5 dpf. (A-A”) Mbp immunolocalization to Mauthner axons in the hindbrain of wild-type animals at 5 dpf (arrows). (B-B”) Mbp immunolocalization to Mauthner axons in the hindbrain of <i>vps11(plt)</i> mutants at 5 dpf (arrows). (C-C”) Mbp immunolocalization in the hindbrain of wild-type animals at 7 dpf. (D-D”) Significant reduction in Mbp immunolocalization in the hindbrain of <i>vps11(plt)</i> mutants at 7 dpf.</p

Brain MRI demonstrated white matter abnormality suggesting leukoencephalopathy in a patient homozygous for <i>VPS11</i>: c.2536T>G (p.C846G).

<p>In patient B at nine months of age, thin corpus callosum (A), T2 diffuse hyperintensity in the peri-Rolandic white matter (B) and posterior occipital white matter region (C) were seen; FLAIR diffuse hyperintensity in the white matter region was also seen (D). At five years of age, diminutive corpus callosum (E), T2 diffuse hyperintensity signal in peri-Rolandic areas (F) and supratentorial white matter, most pronounced within the bilateral parieto-occipital regions (G) were seen; FLAIR diffuse hyperintensity in the white matter region with a mild increase of myelination was seen(H).</p