Severe neonatal hypotonia due to SLC30A5 variant affecting function of ZnT5 zinc transporter

Abstract

The tightly-regulated spatial and temporal distribution of zinc ion concentrations within cellular compartments is controlled by two groups of Zn²⁺ transporters: the 14-member ZIP/SLC39 family, facilitating Zn²⁺ influx into the cytoplasm from the extracellular space or intracellular organelles; and the 10-member ZnT/SLC30 family, mobilizing Zn²⁺ in the opposite direction. Genetic aberrations in most zinc transporters cause human syndromes. Notably, previous studies demonstrated osteopenia and male-specific cardiac death in mice lacking the ZnT5/SLC30A5 zinc transporter, and suggested association of two homozygous frameshift SLC30A5 variants with perinatal mortality in humans, due to hydrops fetalis and hypertrophic cardiomyopathy. We set out to decipher the molecular basis of a severe hypotonia syndrome. Combining homozygosity mapping and exome sequencing studies of consanguineous Bedouin kindred, as well as transfection experiments and zinc monitoring in HEK293 cells, we demonstrate that a bi-allelic in-frame 3bp deletion variant in SLC30A5, deleting isoleucine within the highly conserved cation efflux domain of the encoded ZnT5, results in lower cytosolic zinc concentrations, causing a syndrome of severe non-progressive neonatal axial and limb hypotonia with high-arched palate and respiratory failure. There was no evidence of hydrops fetalis, cardiomyopathy or multi-organ involvement. Affected infants required nasogastric tube or gastrostomy feeding, suffered from various degrees of respiratory compromise and failure to thrive and died in infancy. Thus, a biallelic variant in SLC30A5 (ZnT5), affecting cytosolic zinc concentrations, causes a severe hypotonia syndrome with respiratory insufficiency and failure to thrive, lethal by 1 year of age.

Keywords: SLC30A5; ZnT5; hypotonia; mutation; neurological syndrome.

FIGURE 1

The disease phenotype: (A) family tree of the affected kindred. (B) Chest X‐ray of affected individual V‐3, showing restricted lung expansion (less than five ribs) and upwardly bilaterally displaced diaphragm.

FIGURE 2

The SLC30A5 variant: (A) homozygosity mapping: red marks a single chromosome 5 homozygous locus shared by all affected individuals (Homozygosity‐Mapper). (B) The c.1897_1899delATA SLC30A5 variant (Sanger sequencing). (C) Protein effects of the variant: SMART diagram of SLC30A5 domain organization demonstrating that the deleted isoleucine (red rectangle) is within a conserved cation efflux domain, putatively disrupting its transmembrane helix. Blue rectangles represent transmembrane helix regions (per TMHMM v2.0 software); (D) evolutionary conservation of the mutated sequence: multiple protein sequence alignment of SLC30A5 and its putative homologs in different species (red rectangle highlights the deleted isoleucine).

FIGURE 3

The c.1897_1899delATA ZnT5 variant is associated with lower cytosolic zinc concentration: (A) normalized average traces obtained for one acquisition (n = 29 cells for HEK‐ZnT5‐WT and n = 22 cells for HEK‐ZnT5‐mut). (B) Free zinc concentration revealing significant decreases in cytosolic free zinc levels in cells expressing the mutant form compared to cells expressing wild‐type ZnT‐5 (two independent experiments, n = 97 cells for WT and n = 133 cells for mutant). Values are given as mean ± standard deviation. p = 0.0367. (C) Representative microscopy images for WT and mutant SLC30A5 overexpression in HEK293 cells. A further selection of images is given in Figure S1.