Background The genetic heterogeneity of developmental delay and cognitive impairment is vast. The endocytic network is essential for neural development and synaptic plasticity by regulating the sorting of numerous transmembrane proteins. Disruption of the pathway can lead to neuronal pathology. Endosomal biogenesis relies on two Rab proteins, Rab5 and Rab7, which bind to two hexameric tethering complexes, the endosomal class C core vacuole/endosome tethering complex (CORVET) and the late endosomal/lysosomal homotypic fusion and protein sorting complex (HOPS). Both complexes consist of four core proteins and differ by their specific Rab-binding proteins.
Objectives To identify the molecular basis of a neurological disease, which consists of global developmental stagnation at 3–8 months, increasing appendicular spasticity, truncal hypotonia and acquired microcephaly, with variable seizure disorder, accompanied by thin corpus callosum, paucity of white matter and delayed myelination in eight patients from four unrelated Ashkenazi-Jewish (AJ) families.
Methods Exome analysis, homozygosity mapping and Mup1-GFP transport assay in mutant yeast.
Results Homozygosity for a missense mutation, p.Cys846Gly, in one of the endosomal biogenesis core proteins, VPS11, was identified in all the patients. This was shown to be a founder mutation with a carrier frequency of 0.6% in the AJ population. The homologous yeast mutant had moderate impairment of fusion of the late endosome to the vacuole in Mup1-GFP transport assay.
Conclusions We speculate that in neuronal cells, impairment of fusion of the late endosome to the vacuole would attenuate the degradation of plasma membrane receptors, thereby underlying the progressive neuronal phenotype in our patients. The VPS11 p.Cys846Gly mutation should be added to the AJ carrier screening panel.
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Neural development and the synaptic plasticity required for cognitive development necessitates the dynamic and local remodelling of the protein and lipid content of the neuronal cell surface, including millions of highly specialised synaptic connections.1 Central to controlling the composition of the cell surface is the endocytic network.2 Here, the specific internalisation of transmembrane proteins is balanced by the selective sorting of the internalised proteins and lipids back to the cell surface or to the lysosome for downregulation and degradation. By regulating the sorting of numerous transmembrane proteins, including cell adhesion molecules, signalling receptors and ion channels, the endocytic network regulates the structural and functional remodelling of synapses.1
The endocytic network relies on two Rab proteins for endosomal biogenesis—Rab5, which is required for early endosome fusion, and Rab7, which acts in the fusion of the mature late endosome with the lysosome.2–4 During endosomal maturation, Rab5 and Rab7 bind to two hexameric tethering complexes, the endosomal CORVET (‘class C core vacuole/endosome tethering’) and the late endosomal/lysosomal HOPS (‘homotypic fusion and protein sorting’) complex5–11 (figure 1). In metazoans, CORVET functions in endosome–endosome fusion by binding to Rab5, whereas, at least in yeast, HOPS promotes fusion by interacting with the Rab7-homolog Ypt7.12 ,13 Both CORVET and HOPS consist of four core proteins (collectively named C-proteins), Vps11, Vps16, Vps18 and Vps33, and differ by the Rab5-binding Vps3 and Vps8 in CORVET or Rab7/Ypt7-binding Vps39 and Vps41 in HOPS (figure 1).
We now report on patients with hypomyelination and developmental delay associated with a defect in the C-protein Vps11.
The subjects of this study were eight children from four unrelated Ashkenazi-Jewish (AJ) families (figure 2A) currently 13 months to 21 years old.
All affected children were from normal pregnancies and deliveries with head circumference at birth between +1 and −1 SD. The patients presented with global developmental stagnation between 3 and 8 months of age. Subsequent course was characterised by increasing appendicular spasticity up to grade 3 at Ashworth scale with resultant contractures, truncal hypotonia, which required the use of corset for stabilisation and acquired microcephaly with head circumference eventually at around −2 to −3 SD; however, the youngest patient (C-II-1) did not have microcephaly or descent in percentiles of head circumference at 13 months of age. Peak developmental milestones consisted of independent sitting and 2–3 words of expressive language in two children. No syndromic stigmata were present, and review of systems including clinical immune function has not shown involvement of other organs. No cutaneous or ocular findings were present except in patient C-II-1 who was noted to have subalbinotic retinae. In three children, seizures were noted; however, these were rare, consisted of complex partial seizures from 4–6 months of age, and were easily controlled with antiepileptic medications. Brain MRI disclosed a fairly consistent pattern of thin corpus callosum and paucity of white matter with a normal sequence of myelination, but with delayed onset and rate (figure 3), meeting the definition of hypomyelinating leukodystrophy.14 The most advanced MRI performed at 17 years in patient S-II-2 disclosed cerebellar atrophy as well. The salient features of the patients are summarised in table 1. Exome analysis was opted after routine diagnostic investigations were negative.
Whole exome analysis
Exonic sequences were enriched in the DNA samples of patient Z-II-1, B-II-1 and C-II-1 using SureSelect Human All Exon 50 Mb Kit (Agilent Technologies, Santa Clara, California, USA). Sequences were determined by HiSeq2000 (Illumina, San Diego, California, USA), and 100 bp were read paired end. Reads alignment and variant calling were performed with DNAnexus software (Palo Alto, California, USA) using the default parameters with the human genome assembly hg19 (GRCh37) as a reference. Parental consent was given for DNA studies. The study was performed with the approval of the ethical committees of Hadassah Medical Center and the Ministry of Health. Carrier rate was determined using the variants list of the exome analyses of 3232 unrelated anonymous American AJ.
Yeast strains and plasmids
Genetically modified yeast Saccharomyces cerevisiae was made by homologous recombination of PCR-amplified cassettes. The generated yeast strains are listed in the online supplementary table S1. Vps11 and Vps11 mutant genes were introduced to pRS416 plasmids under the control of the NOP1 promoter for transformation into the vps11Δ background strain. Cells were grown in synthetic medium supplemented with amino acids and 2% glucose at 26°C for 16–20 h to logarithmic phase and an OD600 of 0.6–0.8. Afterwards, the cells were split, washed and resuspended in the medium to an OD600 of 0.2 and further grown for 3–5 h at 26°C. The cultures were then stained with FM4-64 as subsequently described, and pictures were taken.
The vacuolar membrane was stained with 30 μM FM4-64 for 30 min (pulse). Washing with dye-free SDC-URA (Synthetic dextrose complex without uracil) medium and incubation for 1 h (chase) was performed as described.15 Microscopy pictures were taken with the help of an Olympus IX-71 inverted microscope equipped with 100×NA 1.49 objective and a sCMOS camera (PCO). The InsightSSI illumination system was equipped with DAPI-, GFP-, mCherry- and Cy5-filters. SoftWoRx software (Applied Precision) was used to operate the microscope. Z-stacks of 4 μm with 400 nm spacing were used for constrained-iterative deconvolution (SoftwoRx).
Mup1 uptake assay
To analyse Mup1-GFP trafficking, 20 µg/mL methionine was added to the medium subsequent to FM4-64 staining, and cells were incubated for 1 h (chase) at 26°C and 38°C, respectively. After incubation at specific temperatures, the cells were pelleted, resuspended in 20 µL media and subjected to microscopy.
Quantification was performed with FijiImageJ16 and the Cell Counter plugin (developed by Kurt de Vos, University of Sheffield, Academic Neurology). Cells of vps11Δ Mup1-GFP VPS11 (wild type) and vps11Δ Mup1-GFP VPS11 C952G (mutant) both grown at 37°C and with the addition of methionine were counted. The cells were divided into two groups.
The reads obtained from the exome analysis of patients Z-II-1, B-II-1 and C-II-1 were aligned (reference genome Hg19), and variants were called and filtered (detailed in online supplementary table S1). A small number of variants remained for each patient, but given their similar clinical course and neuroradiological findings and their common ethnic background, we assumed a founder mutation and focused on chr11:118951899 T>G, NM_021729.5 c. 2536T>G, p.Cys846Gly (p.C846G) in the VPS11 gene which was present in all the patients in a homozygous form. Among 3232 unrelated anonymous AJs, there were 20 carriers for the p.C846G mutation in the VPS11 gene, indicating a carrier rate of 0.6% in this group (Mark Daly, personal communication). The variant was carried also by 11 of the 60383 individuals whose exome analyses were deposited at the Exome Aggregation Consortium, Cambridge, Massachusetts, USA (URL http://exac.broadinstitute.org; accessed April 2015); no homozygotes were present in these cohorts.
We next searched within our genomic linkage data for patients of AJ origin with white matter disease whose DNA SNP genotyped homozygous regions that encompassed VPS11 and identified a fourth family, family S (figure 2A). The minimal homozygous region shared by all the patients was chr11:118759860–119569987 (47 SNP markers with identical genotype in all the patients). Thus, eight patients from four unrelated families of AJ origin were identified. The homozygous p.C846G mutation in the VPS11 gene segregated with the disease within each family (figure 2A).
As Vps11 is an essential component of HOPS and CORVET complexes, which are required for the transport of cargo along the endocytic pathway, we generated a homologous mutation, C952G, in yeast Vps11. We then expressed Vps11 wild type and the C952G mutant from a centromeric plasmid in the vps11Δ background. Both plasmids complemented the vacuole fragmentation phenotype observed in the non-complemented vps11Δ strain (figure 4A), indicating that HOPS function in vacuole fusion is rescued (figure 4B). To identify subtle defects along the endocytic pathway to the vacuole, we monitored the transport of a methionine permease Mup1 as a GFP-tagged protein from the plasma membrane to the lysosome-like vacuole. Mup1-GFP transport is initiated when cells, which were grown in the absence of methionine, are exposed to excess methionine, and Mup1-GFP is eventually found in the vacuole lumen. We observed that Mup1 uptake assay resulted in 14 of 146 (10%) wild-type cells displaying accumulation of endocytic cargo in vesicular structures that correspond to the late endosome.17 In the corresponding vps11 mutant cells, 20 of 110 (18%) showed this accumulation, suggesting a defect in the fusion of the late endosome to the vacuole.
Finally, we determined the activity of several lysosomal enzymes in the plasma of patient C-II-1 and found slight reduction of β-galactosidase activity 6.1 nmol/h/mL (controls 11.2±2.5), with near-normal activities of total hexosaminidase 388 nmol/h/mL (controls 546±84), and arylsulfatase A 124 nmol/h/mL (controls 131±53).
We report on eight patients with developmental delay, acquired microcephaly and hypomyelination associated with homozygosity for a founder mutation in the VPS11 gene. The mutation, identified independently in four unrelated families, segregated with the disease in each family, resulted in exactly the same neurological phenotype, affected a highly conserved residue throughout evolution, and had a carrier rate of ∼1:160 in people of AJ background.
Vps11 is an essential protein of the endosomal pathway. It is part of HOPS and CORVET, two hexameric tethering complexes, which localise to lysosomes and endosomes, respectively. Human VPS11 is a 921 amino acids protein, which consists of three conserved domains: a predicted WD40 domain (AA 142-292) is likely a part of the N-terminal β-propeller as identified for the Vps18 protein,18 two clathrin domains (AA 417-536, 598-727), which presumably add up as part of a C-terminal alfa-solenoid,19 a RING-finger domain (AA 821-860) and a C-terminal domain (AA 862-909). RING-finger domains are made of six spaced cysteines [C-X(2)-C-X(9,39)-C-X(1,3)-H-X(2,3)-[NCH]-X(2)-C-X(4,48)-C-X(2)-C] and typically bind two zinc atoms. The fourth spaced cysteine residue is the mutated residue in our patients. Loss of Vps11 in zebrafish manifests by oculocutaneous albinism, pericardial oedema, hepatomegaly and premature death underscoring Vps11 role in the fish melanosome maturation.20 Studies in various yeast mutants revealed that Vps11 is an essential component of the endosomal system;21 vps11Δ null mutants exhibited defects in endosomal and vacuolar delivery and in late endosome and vacuole fusion. In contrast, a Vps11 mutant termed Vps11 926Δ that lacks only the RING motif had a relatively selective defect at the docking or fusion at the vacuole, but not at the endosome. Coimmunoprecipitation experiments of the Vps11 mutant revealed that the RING domain is not needed for HOPS assembly.22 It is, therefore, not surprising that the phenotype of the yeast Vps11 C952G mutant, corresponding to the p.C846G mutation in our patients, is relatively mild and restores the vacuole morphology of the vps11Δ background. It also keeps the endolysosomal pathway intact, but causes a mild accumulation of cargo in the late endosome. We speculate that in neuronal cells, this abnormality would attenuate the degradation of plasma membrane receptors, thereby underlying the progressive neuronal phenotype in our patients.
Defects in endosomal maturation are recently being unravelled in neurodegenerative disorders. In the autosomal dominant neuropathy Charcot–Marie–Tooth disease type 2B, mutations in the late endosomal Rab7 GTPase interfere with receptor trafficking, resulting in alteration in intracellular signalling events.23 Specifically, mutant Rab7 delays trafficking of epidermal growth factor receptor (EGFR) to the lysosome, thereby slowing down the process of receptor degradation, causing enhanced EGFR signalling, and increased p38 and Erk1/2 activation.24 Excessive endosome-to-lysosome trafficking in a HOPS-related manner was recently shown to underlie the neurodegeneration in the fly mutant of TBC1D24, which in human beings is associated with epilepsy, deafness and intellectual disability.25–27
Given the requirement for the endosomal network in most body systems, it is unclear why mutated VPS11 has such a selective effect on the brain only. The exclusive organ involvement could be related to specific cargo affinity, to tissue-specific expression of various endosomal network-related proteins or to the slow turnover of neuronal cells, which results in massive accumulations over extended periods of time. The latter explanation would agree with recent findings on Rab7 mutants, which also result in an adult-onset neurodegenerative disease, and similar to our observations on the VPS11 mutant in yeast (figure 4), result in a mild overall defect in transport.23 In summary, recessive p.C846G pathogenic variants in the VPS11 gene are causative of a novel endosomal network defect with clinical features of hypomyelination and marked developmental delay. p.C846G is an AJ founder mutation, and given its considerable carrier frequency in the AJ population (0.6%), we propose that it should be added to the AJ carrier screening panel.
We are grateful to Dr Mark Daly, the Broad Institute, Boston, for carrier rate data and to the patients’ families for their cooperation. This work was partly funded by the DFG (Deutsche Forschunggemeinschaft), UN111/6-1.
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
- Data supplement 1 - Online supplement
SE, FG and CJ contributed equally.
Contributors SE, CJ and DG undertook patient management, collected samples and delineated the phenotype, analysed the data and wrote the paper. FG, JL, Ash and AS a performed the experiments, analysed the data and wrote the paper. SE, CJ, CU and OE conceived and designed the experiments, analysed the data and wrote the paper, undertook patient management, collection of samples and delineation of the phenotype.
Competing interests None declared.
Patient consent Obtained.
Provenance and peer review Not commissioned; externally peer reviewed.