Somatic SLC35A2 mosaicism correlates with clinical findings in epilepsy brain tissue

Abstract

Objective: Many genetic studies of intractable epilepsy in pediatric patients primarily focus on inherited, constitutional genetic deficiencies identified in patient blood. Recently, studies have revealed somatic mosaicism associated with epilepsy in which genetic variants are present only in a subset of brain cells. We hypothesize that tissue-specific, somatic mosaicism represents an important genetic etiology in epilepsy and aim to discover somatic alterations in epilepsy-affected brain tissue.

Methods: We have pursued a research study to identify brain somatic mosaicism, using next-generation sequencing (NGS) technologies, in patients with treatment refractory epilepsy who have undergone surgical resection of affected brain tissue.

Results: We used an integrated combination of NGS techniques and conventional approaches (radiology, histopathology, and electrophysiology) to comprehensively characterize multiple brain regions from a single patient with intractable epilepsy. We present a 3-year-old male patient with West syndrome and intractable tonic seizures in whom we identified a pathogenic frameshift somatic variant in SLC35A2, present at a range of variant allele fractions (4.2%-19.5%) in 12 different brain tissues detected by targeted sequencing. The proportion of the SLC35A2 variant correlated with severity and location of neurophysiology and neuroimaging abnormalities for each tissue.

Conclusions: Our findings support the importance of tissue-based sequencing and highlight a correlation in our patient between SLC35A2 variant allele fractions and the severity of epileptogenic phenotypes in different brain tissues obtained from a grid-based resection of clinically defined epileptogenic regions.

Figure 1. Epileptic spasm and intracranial grid location

(A) Ictal recording of typical epileptic spasms. The spikes and high-frequency activity at ictal onset were broadly distributed in the mesial temporal, frontotemporal, inferior parietal, and occipital contacts (sensitivity: 75 uV, low-frequency filter: 5.3 Hz; high-frequency filter: 500 Hz). (B) Placement of the grid and strips (blue) over the left frontal, temporal, parietal, and occipital lobes. (C) Predominant ictal onset (red) and rapid propagation (orange) over a broad region. (D) Location of the tissue samples and spike quantification. FT = frontotemporal; MT = mesial temporal; Occ = occipital.

Figure 2. Neuroimaging, EEG, and histopathology of differentially affected hippocampus and parietal/occipital lobe

(A) Hippocampus: T2-weighted coronal MRI of the brain demonstrates increased signal within the left temporal white matter with circling of the asymmetric gray-white matter blurring on the left temporal lobe (top left). FDG-PET fused with MRI demonstrates focal area of asymmetrically decreased uptake within the left temporal lobe and hippocampus area; arrows point to the left temporal lobe and left hippocampal region (top right). Intracranial EEG demonstrates high-amplitude spikes (asterisks) abundantly in the hippocampus contacts (bottom left). Histology image at 100×; H&E-stained section from hippocampus shows expected cytoarchitecture (bottom right). (B) Parietal/occipital lobe: T1-weighted coronal MRI of the brain demonstrates no focal abnormality in the left parietal lobe (top left). FDG-PET fused with MRI demonstrates focal area of asymmetrically decreased uptake within the left parietal lobe; arrows point to the left parietal lobe (top right). Intracranial EEG demonstrates high-amplitude spikes (asterisks) only occasionally in the occipital contacts (bottom left). Histology image at 40×; H&E-stained section shows cortical dyslamination with neurons of the occipital neocortex arranged in microcolumns (arrowheads). FDG = fluorodeoxyglucose; H&E = hematoxylin and eosin.

Figure 3. SLC35A2 c.634_635delTC VAFs among different sequencing methods

(A) Comparison of SLC35A2 c.634_635delTC VAFs in 6 brain tissues and blood comparator from the affected patient, using 3 different sequencing techniques. Average sequencing depth at the SLC35A2 variant position for each method is shown in parentheses in legend. The apparent presence of the variant at <1% VAF in the blood sample for the libricon approach is likely due to the high error rate of this PCR-based approach and the high read depth achieved during sequencing. (B) VAFs in 12 brain and 1 matched blood tissue using the NND SLC35A2 targeted sequencing kit. (C) Comparison of VAFs in DNA and RNA from 6 different brain tissues using the NND SLC35A2 targeted sequencing kit. Only nucleic acids from high-quality, frozen tissue samples were used for targeted RNA sequencing. Average sequencing depth for each method is shown in parentheses in legends. NND = NEBNext Direct; VAF = variant allele fraction.