Biallelic Mutations of VAC14 in Pediatric-Onset Neurological Disease

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.

Figure 1

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.

Figure 2

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). (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.

Figure 3

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).

Figure 4

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., , (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.