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. 2016 Jun 9;99(1):188–194. doi: 10.1016/j.ajhg.2016.05.008

Biallelic Mutations of VAC14 in Pediatric-Onset Neurological Disease

Guy M Lenk 1, Krystyna Szymanska 2,3, Grazyna Debska-Vielhaber 4, Malgorzata Rydzanicz 5, Anna Walczak 5, Monika Bekiesinska-Figatowska 7, Stefan Vielhaber 4, Kerstin Hallmann 8, Piotr Stawinski 5,6, Sonja Buehring 9, David A Hsu 10, Wolfram S Kunz 8, Miriam H Meisler 1,∗, Rafal Ploski 5,∗∗
PMCID: PMC5005439  PMID: 27292112

Abstract

In the PI(3,5)P2 biosynthetic complex, the lipid kinase PIKFYVE and the phosphatase FIG4 are bound to the dimeric scaffold protein VAC14, which is composed of multiple heat-repeat domains. Mutations of FIG4 result in the inherited disorders Charcot-Marie-Tooth disease type 4J, Yunis-Varón syndrome, and polymicrogyria with seizures. We here describe inherited variants of VAC14 in two unrelated children with sudden onset of a progressive neurological disorder and regression of developmental milestones. Both children developed impaired movement with dystonia, became nonambulatory and nonverbal, and exhibited striatal abnormalities on MRI. A diagnosis of Leigh syndrome was rejected due to normal lactate profiles. Exome sequencing identified biallelic variants of VAC14 that were inherited from unaffected heterozygous parents in both families. Proband 1 inherited a splice-site variant that results in skipping of exon 13, p.Ile459Profs∗4 (not reported in public databases), and the missense variant p.Trp424Leu (reported in the ExAC database in a single heterozygote). Proband 2 inherited two missense variants in the dimerization domain of VAC14, p.Ala582Ser and p.Ser583Leu, that have not been previously reported. Cultured skin fibroblasts exhibited the accumulation of vacuoles that is characteristic of PI(3,5)P2 deficiency. Vacuolization of fibroblasts was rescued by transfection of wild-type VAC14 cDNA. The similar age of onset and neurological decline in the two unrelated children define a recessive disorder resulting from compound heterozygosity for deleterious variants of VAC14.

Main Text

PI(3,5)P2 is a low-abundance signaling lipid that is localized to the cytoplasmic surface of endolysosomal vesicles.1, 2, 3, 4, 5, 6 The abundance of PI(3,5)P2 is regulated by a ubiquitously expressed protein complex in which the kinase PIKFYVE (MIM: 609414) and the phosphatase FIG4 (MIM: 609390) are bound to the scaffold protein VAC14 (MIM: 604632).7, 8, 9 The physical association of the kinase and phosphatase is thought to permit dynamic regulation of localized concentrations of PI(3,5)P2. Deficiency of PI(3,5)P2 results in enlarged endolysosomal vacuoles, possibly generated by osmotic swelling due to impaired activation of the lysosomal cation channels TRPML1 (MIM: 605248), TPCN1 (MIM: 609666), and TPCN2 (MIM: 612163).10, 11, 12, 13, 14

Neurons are particularly sensitive to reduced abundance of PI(3,5)P2.15 Mutations of FIG4 result in recessive neurological disorders, including the peripheral neuropathy Charcot-Marie-Tooth type 4J (MIM: 611228), the multisystem disorder Yunis-Varón syndrome (MIM: 216340), and polymicrogyria with seizures and psychiatric co-morbidities (MIM: 612691).15, 16, 17, 18 Haploinsufficiency of PIKFYVE results in the benign condition fleck corneal dystrophy (MIM: 121850) with accumulation of intracytoplasmic vesicles.19 Pathogenic mutations of human VAC14, which is located on chromosome 16q22, have not previously been reported.

In mice, mutations of all three of the major proteins in the PI(3,5)P2 biosynthetic complex have been characterized. A spontaneous null allele of Fig4 resulted in reduced abundance of PI(3,5)P2 and extensive neurodegeneration in the CNS and peripheral nervous system (PNS), which was rescued by a neuron-specific transgene.15, 20, 21 Ubiquitous knockout of Pikfyve resulted in pre-implantation lethality,22 a hypomorphic mutation resulted in neonatal lethality,23 and conditional knockout in platelets resulted in impaired lysosome secretion and thrombosis.24 Two mutant alleles of mouse Vac14 have been studied. The Vac14βgeo-null mutation resulted in neonatal lethality and neurodegeneration of the CNS and PNS, similar to what occurs in the Fig4-null mouse.25 The missense mutation p.Leu156Arg prevents binding of VAC14 to PIKFYVE, leading to extensive neurodegeneration and juvenile lethality at 1 month.7

In this report, we describe two children with abrupt onset of progressive neurological disease due to recessive mutations of VAC14. Appropriate informed consent was obtained from all subjects or their parents, and the procedures followed were in accordance with the ethical standards of the responsible committees on human experimentation at Warsaw Medical University, the University of Wisconsin–Madison, and the University of Michigan. All studies including whole-exome sequence and biopsy were performed with parental consent.

The affected individuals were males who developed normally until the onset of rapidly progressive neurological decline without an obvious precipitating cause. Their clinical features are compared in Table 1. At 3 years and 1 month of age, proband 1 developed dystonic gait, pain in the lower extremities, and progressive dystonia of the lower and then the upper extremities. At 5 years of age, he was nonverbal and nonambulatory and had marked hypersalivation, increased muscle tone, and dystonic movements of facial, limb, and trunk muscles (see Supplemental Note). Proband 2 walked at 12 months but developed an abnormal gait at 18 months. At 25 months he was unable to crawl and required assistance to stand. Speech slowed, and he was only able to produce two- or three-word sentences. Chewing and swallowing slowed, with hypersalivation. Details regarding progression and responses to treatment are provided in the Supplemental Note.

Table 1.

Clinical Features of the Two Probands

Proband 1 Proband 2
Family history nonconsanguineous parents with no family history nonconsanguineous parents with no family history
Pregnancy/perinatal period first/normal; caesarean section first/normal
Apgar at birth 10 9–10
Weight at birth 3,900 g 3,600 g
Head circumference normal normal
Gender male male
Psychomotor development normal; sitting at 7 months, walking at 12 months normal; sitting at 6 months, walking at 12 months
First symptoms abnormal gait, loss of walking, dystonic leg movements abnormal gait, loss of walking
Onset abrupt at 3 years abrupt at 18 months
Progression yes yes
Dystonia dystonia; episodes of status dystonicus dystonia
Muscle tone increased tone in trunk and extremities (rigidity) weakness of trunk muscles, increased tone in lower extremities
Involuntary movements dystonic movements of jaw, neck, back, and extremities not observed
Treatments baclofen, l-dopa/carbidopa, benzodiazepines, immunoglobulin, steroids, biperiden hydrochloride, clonidine l-dopa/sinemet
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MRI results (see Figure 1) are described in the text.

Brain MRI for proband 1 at 3 years of age revealed two types of signal alteration (Figures 1A–1D). T2, fluid-attenuated inversion recovery (FLAIR), and bilateral diffusion-weighted hyperintensities were evident in the striatum, and there was susceptibility-weighted hypointensity of the pallidum and substantia nigra that progressed over time. This appearance was not typical of Leigh syndrome, which was also counterindicated by the absence of lactic acid peak on magnetic resonance spectroscopy. Brain MRI for proband 2 at 2 years of age revealed involvement of the striatum with T2 and FLAIR hyperintensities, similar to proband 1 (Figure 1E). There was no change of signal intensity in the basal ganglia on diffusion-weighted imaging (Figure 1F), no hypointensity on susceptibility-weighted imaging in the pallidum (Figure 1G), and a trace of susceptibility-weighted hypointensity in the substantia nigra (Figure 1H). Possible enhancement of nerve roots and caudal equina was observed on MRI of the spine.

Figure 1.

Figure 1

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Striatal Abnormalities Revealed by MRI in Probands 1 and 2

(A) MRI of proband 1 at 3 years of age reveals FLAIR hyperintensity of the caudate nuclei and putamina that form the striatum (arrows).

(B) Increased signal intensity of the striatum on diffusion-weighted imaging.

(C) Hypointensity of the pallidum (arrow) on susceptibility-weighted imaging.

(D) Hypointensity of the substantia nigra (arrow) on susceptibility-weighted sequence.

(E) MRI of proband 2 at 2 years of age also revealed involvement of the striatum, with FLAIR hyperintensity similar to proband 1 (arrows).

(F) Signal intensity in the basal ganglia is not altered on diffusion-weighted imaging.

(G) Signal intensity in the pallidum is not altered on susceptibility-weighted imaging.

(H) Tiny foci of hypointensity in the substantia nigra on susceptibility-weighted imaging.

Clinical exome sequencing of gDNA from peripheral blood of both probands identified biallelic point mutations in VAC14 that were verified by Sanger sequencing and found to be inherited from unaffected heterozygous parents (Figure 2A). In proband 1, the VAC14 (GenBank: NM_018052.3) mutation c.1271G>T in exon 11, resulting in the missense substitution p.Trp424Leu, was paternally inherited, and a G>A nucleotide substitution at the +1 position downstream of exon 13 (c.1528+1G>A, g.70778325C>T [GenBank: NC_000016.9]) that disrupts the consensus splice-donor site for intron 13 was maternally inherited. In proband 2, the inherited missense mutations c.1744G>T and c.1748C>T, affecting adjacent amino acid residues p.Ala582Ser and p.Ser583Leu, were identified (Figure 2A).

Figure 2.

Figure 2

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Inheritance of VAC14 Variants

(A) Recessive inheritance of VAC14 alleles from unaffected heterozygous parents in two pedigrees.

(B) Location of the four inherited variants on the VAC14 cDNA. Exon junctions are shown, and the dimerization domain is shaded in gray.

(C) Model of the PI(3,5)P2 biosynthetic complex (adapted from Alghamdi et al., 2013).26

(D) Evolutionary conservation of the three amino acid residues altered by missense mutations in probands 1 and 2. Aligned VAC14 sequences from GenBank: NP_060522.3 (Homo sapiens), NP_666328.2 (Mus musculus), NP_001025735.1 (Gallus gallus), XP_008119452.1 (Anolis carolinensis), XP_003964762.1 (Takifugu rubripes), and XP_004208946.2 (Hydra vulgaris). Clinical exome sequencing for proband 1 was carried out at Warsaw University with the SureSelect Enrichment kit (Agilent) and 100 bp paired-end sequencing on the HiSeq 1500 Automated Sequencer (Illumina) with a minimum of 10× coverage for 95% of the target and a minimum of 20× coverage for 88%. Clinical exome sequencing for proband 2 was carried out by GeneDx with the Agilent Clinical Research Exome kit with mean depth of coverage of 62×. Variants were verified by Sanger sequencing.

The locations of these variants in the VAC14 cDNA are shown in Figure 2B, and the orientation of VAC14 within the PI(3,5)P2 biosynthetic complex is shown in Figure 2C. The three VAC14 residues with missense mutations are highly conserved during evolution (Figure 2D). These missense substitutions are predicted to be deleterious by the programs PolyPhen-2, SIFT, and MutationTaster (Table S1). p.Trp424Leu was reported in a single heterozygote in the Exome Aggregation Consortium (ExAC) database, which includes sequences from approximately 60,000 control individuals (allele frequency = 0.00001). The splice-site mutation in proband 1 and the missense mutations in proband 2 are not reported in ExAC. The substitutions at Ala582 and Ser583 change the alternation of hydrophilic and hydrophobic residues and result in one allele with adjacent hydrophobic residues (Ala582 and Leu583) and the other allele with adjacent hydrophilic residues (Ser582 and Ser583). These residues are located within the domain required for VAC14 dimerization, which is essential for binding of FIG4,26 and could interfere with association of mutant monomers into stable dimers. We previously reported that loss of VAC14 leads to instability of FIG4.20 Both substitutions are predicted to be pathogenic by PolyPhen-2, SIFT, and MutationTaster (Table S1).27, 28, 29

To evaluate the effect of the splice-site mutation in proband 1, RNA was isolated from cultured fibroblasts and amplified by RT-PCR with a forward primer complementary to the exon 10 and exon 11 junction and a reverse primer from exon 15. Electrophoresis of the RT-PCR products detected the wild-type fragment of 575 bp and an abnormal product of 418 bp present at a much lower level (Figure 3A). The abnormal product contains sequence corresponding to the exon 12 and exon 14 junction rather than the exon 12 and exon 13 junction in the wild-type fragment (Figure 3B), demonstrating that the splice-site mutation in allele 1 results in skipping of exon 13. Given that exon 13 contains 157 bp, deletion of exon 13 alters the reading frame and introduces a premature stop codon, generating the predicted truncated protein p.Ile459Profs∗4. The low abundance of the exon-skipped 418 bp RT-PCR product (Figure 3A) suggested that the transcript with the stop codon might be degraded by nonsense-mediated decay. To directly compare the abundance of the two transcripts, allele 1 with the splice-site mutation encoding wild-type Trp424 in exon 11 and allele 2 with the mutant Leu424 codon, we amplified a cDNA fragment containing exon 11, using the forward primer described above and a reverse primer spanning the junction between exon 12 and exon 13. The sequence chromatogram revealed that the mutated T nucleotide in Leu codon TTG was the major product, whereas the wild-type G nucleotide in Trp codon TGG was not detected (Figure 3C). These data support the prediction that the transcript of allele 2 with the splice-site mutation is mis-spliced and degraded by nonsense-mediated decay.

Figure 3.

Figure 3

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The Splice-Site Mutation in Proband 1 Results in Skipping of Exon 13, Protein Truncation, and Nonsense-Mediated Decay

RNA was prepared with the Trizol Reagent (Life Technologies) and the RNeasy MiniKit (QIAGEN). cDNA was produced from RNA templates with the iScript Select cDNA synthesis kit (Bio-Rad Laboratories) with random primers for reverse transcription and amplified with a forward primer spanning the exon 10 and exon 11 junction (5′-GTC TTC ACT GCA GCC AGC ACT GA-3′) and a reverse primer in exon 15 (5′-AGG ATG TCT GCC ATT GAG TGG AAG-3′).

(A) In addition to the predicted product of 575 bp amplified from control fibroblasts, a low level of an abnormal product of 418 bp was obtained from proband 1’s RNA (arrow).

(B) Sequence of the 575 bp fragment from control fibroblasts (upper) and the 418 bp RT-PCR product from the heterozygous proband (lower). The sequence of the 418 bp product includes a junction between exon 12 and exon 14, demonstrating skipping of exon 13. Ile459 is the first codon that is different in the wild-type versus mutant transcript.

(C) The forward primer spanning the exon 10 and exon 11 junction was used in combination with a reverse primer spanning the exon 12 and exon 13 junction (5′-CTT CAG GAT CAC CTC ATC CGA-3′) to determine the sequence of codon 424. Only transcripts containing the mutant Leu codon were detected, consistent with nonsense-mediated decay of the incorrectly spliced transcript in (A).

Cultured fibroblasts from both probands exhibit the vacuolization phenotype that is characteristic of PI(3,5)P2 deficiency (Figure 4A). To evaluate the role of the VAC14 mutations in this phenotype, we tested the ability of wild-type VAC14 cDNA to rescue vacuolization, as previously described.17, 18 Cells from proband 1 were plated at 30% confluence and co-transfected with wild-type VAC14 cDNA or an empty vector and GFP cDNA as a marker for transfected cells. After 48 hr in culture, we examined cells by fluorescence microscopy to identify transfected cells and by phase-contrast microscopy to assess vacuolization. 80% ± 3% of cells transfected with an empty vector contained six or more vacuoles, but after transfection with VAC14 cDNA, this was reduced to 18% ± 5% (n = 3, mean ± SD, p < 0.0001, unpaired t test [two-tailed]) (Figures 4B–4D). These data demonstrate deficiency of functional VAC14 and support the view that VAC14 deficiency is responsible for the neurological disorder.

Figure 4.

Figure 4

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Vacuolization of Proband Fibroblasts and Rescue by Transfection of VAC14 cDNA

(A) Cultured fibroblasts from probands 1 and 2 accumulate cytoplasmic vacuoles with the characteristic appearance of vacuoles seen in PI(3,5)P2-deficient cells.15, 17, 25

(B) Control transfections of proband 1 fibroblasts with GFP cDNA and an empty vector do not affect the vacuoles.

(C) Transfection with VAC14 cDNA rescues vacuolation of proband 1 fibroblasts.

(D) Quantitation of vacuolization in transfected fibroblasts from proband 1. In three independent experiments, transfection with vector generated 80% ± 3% vacuolated cells, and transfection with wild-type VAC14 generated 18% ± 5% vacuolated cells, mean ± SD, p < 0.0001, unpaired t test (two-tailed). Primary fibroblasts were cultured in DMEM with 1× antibiotic and antimycotic and 15% fetal bovine serum with 25 mM HEPES (pH 7). For transfection, cells were plated at 30% confluence and transfected with 2 ug of each construct with Lipofectamine 3000. Live-cell imaging was carried out on a Leica inverted microscope.

In summary, exome sequencing of two unrelated probands independently identified biallelic mutations of VAC14 as most likely to be responsible for their neurological disease. Cultured fibroblasts from both individuals were found to exhibit the vacuolization phenotype that was previously associated with PI(3,5)P2 deficiency due to mutation of human and mouse FIG4, mutation of mouse Vac14,7, 15, 17, 18, 25, 30 and also chemical inhibitors of the pathway, such as apilimod.31, 32, 33 The mouse and human mutations leading to PI(3,5)P2 deficiency also result in neurological disorders. The vacuolization of cultured fibroblasts, and the rescue by VAC14 cDNA, provides strong evidence for pathogenicity of the VAC14 mutations. The phenotypes of the probands are consistent with the demonstrated role of PI(3,5)P2 in the nervous system. Both individuals have involvement of the striatum on MRI, including T2 and FLAIR hyperintensities, that might be accounted for by in vivo vacuolization.

It is of interest to compare the probands described here with the spectrum of phenotypes associated with mutation of FIG4 encoding another subunit of the widely expressed PI(3,5)P2 biosynthetic complex. Yunis-Varón syndrome is associated with homozygosity for null alleles of FIG4 and results in severe degeneration of the CNS, including extensive neuronal loss, diffuse atrophy, and vacuolization of neurons, muscle, cartilage, heart, macrophages, and osteoblasts.17 In individuals with the less severe Charcot-Marie-Tooth disease type 4J, caused by compound heterozygosity for one null allele and one partial loss-of-function allele, abnormalities are limited to the PNS but result in a severe progressive disorder.16 The probands with VAC14 mutations described here are similar to the individuals with Yunis-Varón in that the CNS is involved. Peripheral nerve function has not yet been assessed in these probands. Involvement of other organs might be anticipated in cases with severe missense or null alleles of VAC14.

The probands with VAC14 mutations are less severely affected than the two recessive mouse Vac14 mutants, the Vac14βgeo-null mouse25 and the Vac14Leu156Arg mouse mutant with a mutation that impairs binding of PIKFYVE.7 The three VAC14 mutant proteins with amino acid substitutions described here thus appear to retain partial activity, and it will be of interest to examine their biochemical function in the future. In contrast to the early lethality of both mouse mutants, the probands experienced 1.5 to 4 years of normal development prior to disease onset. This is reminiscent of individuals with Charcot-Marie-Tooth disease type 4J, who can have decades of normal function prior to the onset of peripheral neuropathy.16 The amino acid residues of VAC14 that are substituted in the probands are highly evolutionarily conserved throughout the metazoa, consistent with strong functional constraint. The detected mutations are very rare or “private” variants, given that only one was previously observed, and that in a single heterozygous individual. The many similarities between the two probands support the view that their abrupt-onset neurological disorder results from compound heterozygosity for inherited deleterious mutations of VAC14. This description of neurological defects associated with mutation of VAC14 will facilitate the interpretation of variants identified in the future by exome sequencing in individuals with similar disorders.

Acknowledgments

We thank Rachel Hollander for technical assistance. This work was supported in part by NIH research grant R01 GM24872 (M.H.M.), National Science Centre grant 2013/11/B/NZ7/04944 (R.P.), and Deutsche Forschungsgemeinschaft grant KU 911/21-1 (W.S.K.).

Published: June 9, 2016

Footnotes

Supplemental Data include a Supplemental Note and one table and can be found with this article online at http://dx.doi.org/10.1016/j.ajhg.2016.05.008.

Contributor Information

Miriam H. Meisler, Email: meislerm@umich.edu.

Rafal Ploski, Email: rploski@wp.pl.

Web Resources

Supplemental Data

Document S1. Supplemental Note and Table S1
mmc1.pdf (107.9KB, pdf)
Document S2. Article plus Supplemental Data
mmc2.pdf (1.1MB, pdf)

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Associated Data

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Supplementary Materials

Document S1. Supplemental Note and Table S1
mmc1.pdf (107.9KB, pdf)
Document S2. Article plus Supplemental Data
mmc2.pdf (1.1MB, pdf)

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