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

Abstract

Objective: To identify whether somatic mutations in SLC35A2 alter N-glycan structures in human brain tissues and cause nonlesional focal epilepsy (NLFE) or mild malformation of cortical development (mMCD).

Methods: Deep whole exome and targeted sequencing analyses were conducted for matched brain and blood tissues from patients with intractable NLFE and patients with mMCD who are negative for mutations in mTOR pathway genes. Furthermore, tissue glyco-capture and nanoLC/mass spectrometry analysis were performed to examine N-glycosylation in affected brain tissue.

Results: Six of the 31 (19.3%) study patients exhibited brain-only mutations in SLC35A2 (mostly nonsense and splicing site mutations) encoding a uridine diphosphate (UDP)-galactose transporter. Glycome analysis revealed the presence of an aberrant N-glycan series, including high degrees of N-acetylglucosamine, in brain tissues with SLC35A2 mutations.

Conclusion: Our study suggests that brain somatic mutations in SLC35A2 cause intractable focal epilepsy with NLFE or mMCD via aberrant N-glycosylation in the affected brain.

Figure 1. Representative radiologic and pathologic images of patients with brain somatic mutations in SLC35A2

(A) Preoperative and postoperative brain MRI T2-weighted images from patients EPI219 and LGS150 with brain somatic mutations in SLC25A2. These T2-weighted images demonstrate no remarkable findings in the brain parenchyma, including the temporal lobe. Yellow arrowhead: putative regions of epileptic focus. (B) Histopathologic images from H & E staining and immunohistochemical (IHC) staining from EPI219 (upper panels) and LGS150 (lower panels) brain tissues. Black arrowheads: scattered neuron in white matter. Scale bars, 40 μm in H & E staining and 200 μm in IHC staining for NeuN, a neuronal marker (C) Capture image from integrative genomic viewer (IGV) (upper panels), showing the results of site-specific amplicon sequencing. Schematic tables (lower panels) showing the number of sequence reads counted as mutated or reference sequences, as well as the VAFs of mutated alleles. Mut: mutation, Ref: reference.

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.