Mutations in SLC30A10 cause parkinsonism and dystonia with hypermanganesemia, polycythemia, and chronic liver disease

Abstract

Manganese is essential for several metabolic pathways but becomes toxic in excessive amounts. Manganese levels in the body are therefore tightly regulated, but the responsible protein(s) remain incompletely known. We studied two consanguineous families with neurologic disorders including juvenile-onset dystonia, adult-onset parkinsonism, severe hypermanganesemia, polycythemia, and chronic hepatic disease, including steatosis and cirrhosis. We localized the genetic defect by homozygosity mapping and then identified two different homozygous frameshift SLC30A10 mutations, segregating with disease. SLC30A10 is highly expressed in the liver and brain, including in the basal ganglia. Its encoded protein belongs to a large family of membrane transporters, mediating the efflux of divalent cations from the cytosol. We show the localization of SLC30A10 in normal human liver and nervous system, and its depletion in liver from one affected individual. Our in silico analyses suggest that SLC30A10 possesses substrate specificity different from its closest (zinc-transporting) homologs. We also show that the expression of SLC30A10 and the levels of the encoded protein are markedly induced by manganese in vitro. The phenotype associated with SLC30A10 mutations is broad, including neurologic, hepatic, and hematologic disturbances. Intrafamilial phenotypic variability is also present. Chelation therapy can normalize the manganesemia, leading to marked clinical improvements. In conclusion, we show that SLC30A10 mutations cause a treatable recessive disease with pleomorphic phenotype, and provide compelling evidence that SLC30A10 plays a pivotal role in manganese transport. This work has broad implications for understanding of the manganese biology and pathophysiology in multiple human organs.

Figure 1

Clinical and Neuroimaging Findings in Individuals with SLC30A10 Mutations (A and G) Pedigrees of the Italian and the Dutch families. Black symbols denote affected individuals. The proband is indicated with an arrow. (B–F) Brain MRI images in the individuals from the Italian family. In both brothers marked T1 hyperintensities, typically seen in persons with manganese intoxication, are present in MTC T1-weighted images in the globus pallidus, putamen, caudate nucleus, midbrain, and cerebellum, bilaterally (axial [B–C] and sagittal [E–F] images in individual SIE-03 [B and E] and SIE-02 [C and F]. In (D), a 3DFFET1-weighted, coronal oblique multiplanar reconstruction image shows hyperintensity along the cerebello-rubro-thalamic pathways in individual SIE-03. (H and I) Brain MRI images in the individuals from the Dutch family. In individual NIJ005, marked hyperintensities are present in T1-weighted images in the globus pallidus, bilaterally (coronal image [H]); in individual NIJ010, T1-weighted images obtained around the time the manganese blood levels had returned at almost normal levels showed no obvious abnormalities (axial image shown in [I]).

Figure 2

Homozygosity Mapping and Detection of SLC30A10 Mutations (A) Genome-wide SNP arrays scan defines one large homozygous region shared by the affected individuals from the Italian and the Dutch families on chromosomal region 1q41-q42. (B) Genomic organization of SLC30A10. (C) Electropherogram showing the SLC30A10 mutations present in the Dutch affected individuals NIJ005 and NIJ010. (D) Electropherogram showing the SLC30A10 mutation present in the Italian affected individuals SIE-02 and SIE-03.

Figure 3

Localization of SLC30A10 in the Liver and Nervous System Immunohistochemistry was performed with an anti-SLC30A10 antibody (Santa Cruz); the images shown are from normal liver tissue (A and C); a liver biopsy from the NIJ012 affected individual (B and D); neurons from globus pallidus (E and F) and spinal cord (G) of a normal donor. In the liver, immunoreactivity is present in the normal hepatocytes (A) and normal bile ducts (C), but absent in the tissue from the affected individual (B and D). The scale bars represent 16.5 μm for all panels.

Figure 4

In Silico Analysis of SLC30A10 and In Vitro Expression Studies (A) Schematic topology of SLC30A10. The predicted effects of the disease-causing mutations are depicted. (B) Alignment of the SLC30A10 and the SLC30A1 homologs. (C) The expression of SLC30A10 is induced in hepatocellular carcinoma cells (HepG2) after exposure to sublethal concentrations of manganese. The cDNA levels of the target gene (SLC30A10) and those of an unrelated gene (YWHAZ) are compared to those of a reference gene (TBP). Two assays are performed for SLC30A10 (Q1 and Q2) yielding similar results. The data shown represent means ± standard error of the mean of experiments performed in triplicates. The statistical analysis that used TBP/SLC30A10 and TBP/YWHAZ ΔCt values from each sample was done with one-way ANOVA (Tukey HSD test). ^∗∗∗p < 0.001 (D) Immunoblotting analysis of HepG2 cells shows increased abundance of a band of the predicted size of SLC30A10 after exposure of the cells to sublethal concentrations of MnCl₂. Actin is used as a loading control.