N-glycome analysis detects dysglycosylation missed by conventional methods in SLC39A8 deficiency

Abstract

Congenital disorders of glycosylation (CDG) are a growing group of inborn metabolic disorders with multiorgan presentation. SLC39A8-CDG is a severe subtype caused by biallelic mutations in the manganese transporter SLC39A8, reducing levels of this essential cofactor for many enzymes including glycosyltransferases. The current diagnostic standard for disorders of N-glycosylation is the analysis of serum transferrin. Exome and Sanger sequencing were performed in two patients with severe neurodevelopmental phenotypes suggestive of CDG. Transferrin glycosylation was analyzed by high-performance liquid chromatography (HPLC) and isoelectric focusing in addition to comprehensive N-glycome analysis using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS). Atomic absorption spectroscopy was used to quantify whole blood manganese levels. Both patients presented with a severe, multisystem disorder, and a complex neurological phenotype. Magnetic resonance imaging (MRI) revealed a Leigh-like syndrome with bilateral T2 hyperintensities of the basal ganglia. In patient 1, exome sequencing identified the previously undescribed homozygous variant c.608T>C [p.F203S] in SLC39A8. Patient 2 was found to be homozygous for c.112G>C [p.G38R]. Both individuals showed a reduction of whole blood manganese, though transferrin glycosylation was normal. N-glycome using MALDI-TOF MS identified an increase of the asialo-agalactosylated precursor N-glycan A2G1S1 and a decrease in bisected structures. In addition, analysis of heterozygous CDG-allele carriers identified similar but less severe glycosylation changes. Despite its reliance as a clinical gold standard, analysis of transferrin glycosylation cannot be categorically used to rule out SLC39A8-CDG. These results emphasize that SLC39A8-CDG presents as a spectrum of dysregulated glycosylation, and MS is an important tool for identifying deficiencies not detected by conventional methods.

Keywords: MALDI-TOF MS; SLC39A8; congenital disorders of glycosylation; glycosylation; manganese.

Figure 1.

Leigh-like lesions of the basal ganglia in SLC39A8-CDG. Cranial MRI performed in both patients revealed symmetric bilateral T2 hyperintense lesion. In patient 1, pronounced symmetric bilateral hyperintensities, especially of the putamen were noted along with volume reduction of the entire basal ganglia (A). Similarly, patient 2 showed T2 hyperintense lesions of the putamen and caudate nucleus along with abnormal perfusion restrictions (B, C).

Figure 2.

Normal glycosylation pattern of serum transferrin in two cases of SLC39A8-CDG. Conventional glycosylation analysis of serum transferrin of probands in comparison to a wildtype control (−) and PMM2-CDG (+) using isoelectric focusing (IEF). Numbers indicate the transferrin isoforms carrying the respective amount of sialic acid residues. No indication of the type II dysglycosylation pattern previously observed in SLC39A8-CDG can be seen in either patient (P1, P2) or their heterozygous parent carriers (P1.1, P1.2, P2.1, P2.2). A small elevation of trisialo-transferrin was noted in a heterozygous carrier of the F203S variant.

Figure 3.

N-glycome analysis detects dysglycosylation in samples from patients with homozygous F203S and G38R mutations with normal transferrin. N-glycans from serum samples were analyzed by MALDI-TOF MS. Results are presented as relative abundance of glycans within the full spectrum. The precursor glycan A2G1S1 (m/z 2227) showed a significant increase in the newly identified SLC39A8-CDG patients (P1 and P2) and historical SLC39A8-CDG cases (subject A and B presented in Park et al. 2015) when compared to heterozygous carriers (p < 0.05) and reference controls (p < 0.01) (A). A2G1S1 abundance between heterozygous carriers and controls was approaching statistical significance (p 0.054). The abundance of bisected glycans was significantly reduced in SLC39A8-CDG patients compared to reference controls (p < 0.001) or heterozygous carriers (p < 0.05) (B). In contrast, no significant difference in the abundance of fucosylated glycan species could be observed (C), whereas sialylated glycans allowed a differentiation between SLC39A8-CDG cases and both controls (p < 0.01) and heterozygous carriers (p < 0.05). (n.s.: not significant, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, error bars signify standard error of mean).

Figure 4.

Manganese deficiency is the key driver of pathogenesis in SLC39A8 deficiency. The defective ion channel leads to intracellular manganese depletion in the Golgi apparatus and mitochondria. (A) In the Golgi apparatus, manganese dependent glycosyltransferases exhibit decreased activity due to lack of their obligatory cofactor. Hypogalactosylation and decreased bisection are a core finding in SLC39A8-CDG. (B) Mitochondrial dysfunction is likely mediated by impaired function of manganese superoxide dismutase (SOD2), likely contributing to a clinical presentation suggestive of mitochondrial disease including T2-hyperintense “Leigh-like” lesions of the basal ganglia. The glycan depiction in this figure follows the current international symbol nomenclature for glycans. Figure created with BioRender.com.