SLC39A8 Deficiency: A Disorder of Manganese Transport and Glycosylation

Abstract

SLC39A8 is a membrane transporter responsible for manganese uptake into the cell. Via whole-exome sequencing, we studied a child that presented with cranial asymmetry, severe infantile spasms with hypsarrhythmia, and dysproportionate dwarfism. Analysis of transferrin glycosylation revealed severe dysglycosylation corresponding to a type II congenital disorder of glycosylation (CDG) and the blood manganese levels were below the detection limit. The variants c.112G>C (p.Gly38Arg) and c.1019T>A (p.Ile340Asn) were identified in SLC39A8. A second individual with the variants c.97G>A (p.Val33Met) and c.1004G>C (p.Ser335Thr) on the paternal allele and c.610G>T (p.Gly204Cys) on the maternal allele was identified among a group of unresolved case subjects with CDG. These data demonstrate that variants in SLC39A8 impair the function of manganese-dependent enzymes, most notably β-1,4-galactosyltransferase, a Golgi enzyme essential for biosynthesis of the carbohydrate part of glycoproteins. Impaired galactosylation leads to a severe disorder with deformed skull, severe seizures, short limbs, profound psychomotor retardation, and hearing loss. Oral galactose supplementation is a treatment option and results in complete normalization of glycosylation. SLC39A8 deficiency links a trace element deficiency with inherited glycosylation disorders.

Figure 1

Clinical Phenotype of SLC39A8 Deficiency (A) Three-dimensional reconstruction of cranial CT images of individual A at age 4 months demonstrates cranial asymmetry due to premature synostoses of coronary and lambdoid sutures. Note the lacunar skull presenting with regions of apparent thinning on right-hand side of the cranium. Movie S1 shows skull reconstruction. (B) Cranial MRI at age 6 months revealed asymmetry of the brain with cerebral atrophy of the left hemisphere. The ventricles are enlarged, especially on the left side of the brain. A subdural hygroma is also present. (C) Photographs of individual A at age 9 months demonstrate several dysmorphic features: divergent strabismus, distinct cranial malformation with asymmetry of the skull, flat face, and low-set ears. (D) Dysproportionate short stature with short limbs, especially of the lower extremities.

Figure 2

Schematic Presentation of Transferrin Glycosylation and Glycosylation Profile of Individual A (A) Transferrin is glycosylated at two asparagine (Asn) residues. The attached glycans consist of N-acetylglucosamine (blue square), mannose (green circle), galactose (yellow circle), and sialic acid (purple rhomboid). Terminal sialic acid residues are negatively charged, so that truncation or loss of a glycan side chain results in an overall change in charge of the transferrin molecule. The main transferrin species in healthy individuals is tetrasialo-transferrin, having four terminal sialic acids. (B) The glycosylation profile of individual A is a type II CDG pattern with increased amounts of trisialo-, disialo-, monosialo-, and asialo-transferrin.

Figure 3

Changes in N-Glycosylation with Galactose Therapy (A) Transferrin isoforms were quantitated by HPLC from serum samples and expressed as percentage of total; during galactose supplementation, normalization of glycosylation is seen. Galactose was given on day 1–29, interrupted from day 30 to 43, and then reinstated. Note the steep decline in tetrasialo-transferrin in the short interval between the two therapy intervals. Tetrasialo-transferrin reached stable values within the normal range approximately 2 months after initiation of the second therapy interval. (B) ESI-TOF mass spectra, before and after 120 days of therapy, identifies the partially defective N-glycans attached to serum transferrin. Whereas the mass spectrum before therapy identified mostly transferrin isoforms with defective galactosylation, 120 days of therapy resulted in a normal glycosylation profile with correct galactosylation. The different N-glycan species are indicated below; only one (of sometimes several) possible structures is depicted.

Figure 4

Overview of the Identified Variants and Their Effect on Glycosylation (A) High-performance liquid chromatography (HPLC) spectra of serum transferrin show an increase in trisialo-transferrin for all affected individuals, when compared to those of a healthy control. The most severe hypoglycosylation is found in the person carrying the p.[Gly38Arg];[p.Ile340Asn] double variant. (B) Isolectric focusing (IEF) of serum transferrin from three of the individuals described by Boycott et al. in this issue (B2, D4, D5) carrying only the homozygous p.Gly38Arg variant, and the two individuals described herein with variants p.[Val33Met; p.Ser335Thr];[p.Gly204Cys] and p.[Gly38Arg];[p.Ile340Asn], respectively. IEF shows increased trisialo-transferrin in all affected persons. (C) Alignment of the five identified variants in SLC39A8 with amino acid exchanges highlighted in yellow. Indicated in red are amino acids that are conserved throughout all analyzed species. Blue denotes amino acids with inter-species variation. The number indicates the position of the exchanged amino acid. All identified variants result in an amino-acid exchange in highly conserved regions of the ZIP8 protein encoded by SLC39A8.