Dados do Trabalho

Título

Transcriptome analyses of the white matter of focal cortical dysplasia type II: GPNMB emerges as a potential novel specific marker for balloon cells in FCD IIb

Resumo

Focal cortical dysplasia (FCD) is a frequent cause of pharmacoresistant epilepsy. Based on the 2011 International League Against Epilepsy classification, FCD type II is characterized by dysmorphic neurons (IIa and IIb) and may be associated with balloon cells (IIb). Although the epileptogenic zone of such lesions seems to be mainly cortical, alterations in the white matter (WM) may also play a role in seizure occurrence. Thus, the identification of molecular changes in the WM could contribute with characterization of pathophysiology mechanisms in FCD type II. Here, we aimed to perform a high-throughput molecular characterization of the WM of FCD IIa and IIb and to validate potential biomarkers of these lesions at the protein level. This was a multicentric retrospective study, in which we assessed the transcriptome profiles (RNA-Seq; Illumina HiSeq 2500 platform) of fresh-frozen samples from patients with histopathological diagnosis of frontal FCD (IIa (n=7) or IIb (n=7)) and of frontal neocortex from autopsied individuals without epilepsy (controls, n=4). To avoid biases from tissue homogenates, we performed macrodissections of all samples and separately sequenced the white matters. The validation was performed in formalin-fixed, paraffin embedded tissue from FCD (IIa (n=4) or IIb (n=10)) and autopsy controls (n=8). In this setting, both qualitative and quantitative protein analyses were done with immunohistochemistry. A total of 2 and 24 transcripts were differentially expressed between FCD IIa and controls and FCD IIb and controls, respectively. Complementarily, 4 transcripts were differentially expressed between FCD IIa and IIb. Of note, GPNMB was consistently upregulated exclusively in FCD IIb, compared to both controls and FCD IIa. This marker was, thus, selected for validation. Immunohistochemical reactions detected virtually no GPNMB-immunopositive cells in controls and FCD IIa lesions. Interestingly, a positivity for this marker was found in all FCD IIb samples, and exclusively in balloon cells. The upregulation of GPNMB seen at the transcriptome was quantitatively validated at the protein level, after pixel counting (p=0.002 for FCD IIb vs control and p=0.01 for FCD IIb vs FCD IIa, Kruskal-Wallis test followed by Dunn’s test). We describe and validate GPNMB as a potential specific biomarker for FCD IIb lesions, which may guide future studies to better understand the pathophysiology of these lesions.