Variants in the zinc transporter TMEM163 cause a hypomyelinating leukodystrophy

Abstract

Hypomyelinating leukodystrophies comprise a subclass of genetic disorders with deficient myelination of the CNS white matter. Here we report four unrelated families with a hypomyelinating leukodystrophy phenotype harbouring variants in TMEM163 (NM_030923.5). The initial clinical presentation resembled Pelizaeus-Merzbacher disease with congenital nystagmus, hypotonia, delayed global development and neuroimaging findings suggestive of significant and diffuse hypomyelination. Genomic testing identified three distinct heterozygous missense variants in TMEM163 with two unrelated individuals sharing the same de novo variant. TMEM163 is highly expressed in the CNS particularly in newly myelinating oligodendrocytes and was recently revealed to function as a zinc efflux transporter. All the variants identified lie in highly conserved residues in the cytoplasmic domain of the protein, and functional in vitro analysis of the mutant protein demonstrated significant impairment in the ability to efflux zinc out of the cell. Expression of the mutant proteins in an oligodendroglial cell line resulted in substantially reduced mRNA expression of key myelin genes, reduced branching and increased cell death. Our findings indicate that variants in TMEM163 cause a hypomyelinating leukodystrophy and uncover a novel role for zinc homeostasis in oligodendrocyte development and myelin formation.

Keywords: TMEM163; genomic testing; hypomyelinating leukodystrophies; oligodendrocytes.

Figure 1

Pedigree, variant conservation and protein structure. (A) Pedigree of all four families. (B) Clustal alignment of TMEM163 protein from 11 vertebrate species. The residues altered in the presented individuals are marked in red. (C) Location of the three identified variants shown on a schematic representation of TMEM163 protein.

Figure 2

MRIs findings at different time intervals. Axial T₂-weighted and sagittal T₁-weighted images of S1–S5. S1–S4 show diffuse hyperintense signal in the supra- and infratentorial white matter suggesting hypomyelination. At the age of 7 years, S1 showed minimal progression of myelination in the middle cerebellar peduncles, the cerebellar white matter and the optic radiation (white arrow) and the corpus callosum was thin. MRI of S2 at age 5 years demonstrated improved myelination in the thalamus, cerebral peduncles, pons and medulla. MRI of S5 at age 29 years showed only a non-specific T₂-hyperintense signal abnormality in the parietal periventricular white matter (thick white arrow) and subtly elevated T₂ signal of the peridentate white matter including the hilus of the dentate nucleus (black arrows). Y = years; M = months; W = weeks.

Figure 3

TMEM163 mutants exhibit functional defects in Zn efflux and oligodendrocyte function and survival. (A) Summary of the zinc efflux data obtained using HEK-293 cells electroporated with wild-type (WT) and mutant TMEM163-mCherry constructs. Each point is a biological replicate from 3–4 independent trials. During each trial zinc efflux values were normalized to average values of flux reported in WT samples in each set. Zn efflux is significantly reduced in HEK293 cells transfected with mutant constructs when compared to WT. ***P < 0.001, ****P < 0.0001 using ANOVA with Dunnett’s test corrected for multiple testing. (B) Expression of myelin genes Plp1, Mbp and Cnp in oli-neu cells electroporated with WT and mutant TMEM163-mCherry constructs. Expression of individual genes was analysed by real-time PCR and normalized to Actb as an internal control and plotted as a ratio of WT expression. Expression was significantly reduced for all three genes in cells with the mutant plasmids when compared to WT. Error bars are standard error of the mean, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 analysed by Student's t-test. (C) Overlay epifluorescence images of the oli-neu cells transfected with TMEM163-mCherry (red channel) constructs and stained with β-tubulin (green channel). β-Tubulin marks the cytoplasmic processes in oli-neu cells (scale = 50 µM). Left panel is from the WT construct while right panel is from the L76P mutant construct. (D) Percentage of the cells with more than two processes. Error bars are standard error of the mean from more than 200 cells counted for each replicate, ***P < 0.001, calculated by Student’s t-test compared to control. (E) Fraction of apoptotic oli-neu cells measured as the mean ratio of caspase positive cells to number of transfected cells from >200 cells. ***P < 0.001, calculated by Student’s t-test compared to WT. (F) Similar experiment in HEK293 cells shows no significant difference in fraction of apoptotic cells between WT and mutants.