Brain somatic mutations in SLC35A2 cause intractable epilepsy with aberrant N-glycosylation

Results

Although no pathogenic or recurrent germline mutations were identified in genes potentially linked to intractable focal epilepsy using targeted hybrid capture sequencing (table e-5 and table e-6, links.lww.com/NXG/A124), we surprisingly found that brain somatic mutations in SLC35A2 presented as stop-gained variants, SLC35A2 c.589C > T (p.Gln197*) and c.760 G > T (p.Glu254*) on the X chromosome, in 2 of the 13 patients, respectively (table and table e-4). Both of these 2 patients (LGS150 and EPI219) were male. Their VAFs were 23% for p.Gln197* and 16% for p.Glu254*. Histopathologic analysis of mutation-carrying brain tissues revealed the absence of dysmorphic neurons and balloon cells, as well as any neuronal abnormality other than scattered neurons, which is consistent with mMCD (figure 1, A and B). We confirmed that the 2 variants were detected only in the brain with a similar mutational burden and not in peripheral tissue by site-specific amplicon sequencing (figure 1, C). According to calculated probability values, none of the peripheral tissue sequencing data were statistically significant (table e-7).

In the additional 18 patients with intractable focal epilepsy, deep targeted sequencing of SLC35A2 in matched brain-blood samples revealed brain somatic mutations in SLC35A2, c.703T > G (p.Asn235Gln), c.502 G > A (p.Gln168*), c.553 G > A (p.Gln185*), and chrX:48763821C > A (splice acceptor site mutation) in 4 patients (3 NLFE and 1 mMCD subjects), respectively (figure e-2A, e-2B, links.lww.com/NXG/A124 and table e-1, links.lww.com/NXG/A124). Two of the 4 patients were male. Site-specific amplicon sequencing in matched blood samples from these patients confirmed the absence of the identified somatic variants in peripheral genomic DNA (figure e-2C). The VAFs of these identified mutations ranged from 5% to 18%. Calculating the probability values for amplicon sequencing data for matched peripheral tissue revealed nothing of statistical significance (table e-7). None of the identified variants in SLC35A2 were reported in the Exome Aggregate Consortium database.¹⁸ The mutations were primarily nonsense and splicing site mutations, likely acting as loss-of-function mutations (figure 2A).

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Figure 2 Patient brain tissues with somatic mutations in SLC35A2 encoding a UDP-galactose transporter exhibiting aberrant N-glycosylation

(A) Schematic figure showing brain somatic mutations in SLC35A2 identified in this study. Red star: locations of each identified mutation (B) Extracted compound chromatograms (ECCs) of N-glycans from brain tissues. EPI219 and LGS150: subjects carrying somatic mutations in SLC35A2. Control EPI166: patient with intractable focal epilepsy confirmed to have no specific somatic or germline mutations in deep WES. Control MET886 and MET344: specimens from the tumor-free margin of individuals with a metastatic tumor as part of a planned resection. These specimens were pathologically confirmed as normal brain tissue. The ECCs were color coded according to N-glycosylation types: blue for complex-type glycans containing galactose residues, red for truncated-type glycans, green for high mannose glycans, sky blue for hybrid-type glycans, and pink for the glycans involving high degrees of HexNAc residues. Pink round rectangle square: N-glycan structures showing high degrees of N-acetylglucosamine (HexNAc), such as Hex₃HexNAc₇Fuc₁ and Hex₃HexNAc₈Fuc₁ (C) Representative CID MS/MS spectrum of aberrant N-glycan Hex₃HexNAc₇Fuc₁ in the positive ion detection mode. Almost all fragment ions were single-protonated ions [M + H]+; others are indicated as a superscript. Pink square: Hex₃HexNAc₇Fuc₁ glycan, identified by collision-induced dissociation MS/MS, representing the ion at M/Z 1036.90.

To rule out the possible involvement of somatic mutations in the mTOR pathway in patients with mutations in SLC35A2, we performed deep targeted hybrid capture sequencing (mean depth, 812X) of 10 known mTOR pathway genes, in which no somatic mutations were discovered. In addition, we performed coimmunostaining for phosphorylated S6 and NeuN, a neuronal marker, in freshly frozen brain tissues from 2 SLC35A2 mutation-carrying patients and 1 MTOR p.A1459D mutation-carrying patient. The immunostaining results revealed marked decreases in the numbers of phosphorylated S6 stained neuron cells in patients carrying the SLC35A2 mutation (figures e-4A and e-4B, links.lww.com/NXG/A124).

SLC35A2 encodes a member of the nucleotide-sugar transporter family¹⁹ (figure 2A). The encoded protein transports uridine diphosphate (UDP)-galactose from the cytosol into Golgi vesicles, where it serves as a glycosyl donor for the generation of glycans and plays a crucial role in the galactosylation of N-glycans.²⁰ To examine N-glycosylation profiles in affected brain tissue, we performed TGC and nanoLC/MS analysis in tissues from 2 patients (EPI219 and LGS150) with SLC35A2 c.589C > T (p.Gln197*) and c.760 G > T (p.Glu254*), respectively. The TGC process comprised selective enrichment steps that were critical to detecting trace amounts of glycans from brain tissue: (1) Plasma membrane extraction using ultracentrifugation isolated membrane fractions, including glycoproteins, from homogenized tissue samples. After enzymatic N-glycan release, (2) solid-phase extraction based on porous graphitized carbon was used to remove detergents (e.g., buffer chemicals and nonglycan species) and to capture solely N-glycans.^17,21 The concentrated N-glycans from each brain tissue could be determined by high sensitive nanoLC/MS analysis. The technical reproducibility and sensitivity of the combined platform of TGC and nanoLC/MS have already been demonstrated in a previous study on brain glycomes at the microgram level. Using the proven method, we found that mutation-carrying samples had less galactosylation (associated with truncated glycans without galactose residues) than control brain samples, which is consistent with a previous study¹⁹(figure 2B). In particular, we found that mutation-carrying samples had less galactosylation than control brain samples, which is also consistent with a previous study²²(figure 2B). N-glycan structures were unique, showing high degrees of N-acetylglucosamine (HexNAc), such as Hex₃HexNAc₇Fuc₁ and Hex₃HexNAc₈Fuc₁, on high sensitive LC/MS analysis (figure 2, B and C). The glycan representing the ion at m/z 1036.90, corresponding to [Hex₃HexNAc₇Fuc₁+2H]²⁺, was identified by collision-induced dissociation MS/MS (figure 2C). In sequence, the initial loss of HexNAc residues was clearly observed from the parent ion, indicating that 5 HexNAc residues were linked to the N-glycan core (i.e., Man₃GlcNAc₂). These results suggest that somatic loss-of-function mutations in SLC35A2 lead to aberrant N-glycan patterns of Hex₃HexNAc₇Fuc₁ in patient brain tissues.