Biallelic variants in PIKFYVE cause a hypomyelinating leukodystrophy with endolysosomal dysfunction

The PIKfyve-FIG4-VAC14 lipid kinase complex generates phosphoinositide PI(3,5)P2. Pathogenic variants in VAC14 and FIG4 cause neurodevelopmental disorders and PIKFYVE has been linked to autosomal dominant fleck corneal dystrophy. We searched for the underlyging, genetic cause of a neurodevelopmental disorder in five affected individuals from three families using exome- or genome sequencing. Cranial MRI images and clinical phenotypes were analyzed. Size, number and acidity of cellular vacuoles were investigated in patient-derived fibroblasts from family 1 using LysoSensor acidophilic probes. Levels of PI(3,5)P2 were quantified in patient-derived fibroblasts from family 1 using high-performance liquid chromatography-tandem mass spectrometry. Pikfyve-mutant zebrafish larvae were injected with wildtype-mRNA or mutant mRNA harboring the PIKFYVE missense variant identified in family 3. Pikfyve-mutant zebrafish larvae were treated with metformin, bafilomycin A1, or rapamycin. We identified biallelic PIKFYVE variants in five affected individuals with hypomyelinating leukodystrophy, brain hyperintensities of inferior olives, thalami, long tracts, and optic nerves. Clinically, they presented with intellectual disability, spasticity, muscular weakness, and nystagmus. Additional features included feeding difficulties, auditory dysfunction, insulin resistance, microcephaly, peripheral neuropathy, and mild creatine kinase elevations. We detected four distinct PIKFYVE variants: three loss-of-function variants (family 1 & 2), and one p.Arg1156Pro missense variant (family 3). The pathogenicity of the p.Arg1156Pro variant was confirmed by phenotypic rescue of pikfyve-mutant zebrafish larvae injected with wildtype-mRNA but not with p.Arg1156Pro mRNA. Consistent with the role of PIKfyve in maintaining endolysosomal homeostasis, patient-derived fibroblasts of family 1 accumulated enlarged, hyperacidic vacuoles and showed a reduced PI(3,5)P2 production upon hyperosmotic stimulation. Exposing pikfyve-mutant zebrafisch larvae to the mTORC1 inhibitor and TRPML1 activator rapamycin attenuated cellular vacuolization, highlighting potential therapeutic approaches. Our findings establish PIKFYVE deficiency as a novel cause of hypomyelinating leukodystrophy and endolysosomal dysfunction.