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CODING AND SMALL NON-CODING TRANSCRIPTIONAL LANDSCAPE OF TSC
Introduction
Tuberous Sclerosis Complex (TSC) is a genetic disorder caused by mutations in either TSC1 or TSC2 genes, leading to the development of benign lesions/hamartomas in mul- tiple organs1, 2. TSC often compromises the central nervous system resulting in complex neurological manifestations consisting of varying combinations of neurodevelopmen- tal delay (including autism), various psychiatric disorders and severe epilepsy3-5. Cortical tubers, a form of focal cortical dysplasia, are thought to be major contributors to these disabling neurological manifestations in TSC and are targeted for surgical resection in TSC patients with pharmacologically intractable epilepsy6-8. Although the current surgical and pharmacological management of seizures in TSC often provide significant benefits5, 7, 9, 10, there is further need for a better understanding of the molecular and physiological basis of the neurological manifestations seen in TSC11.
Previous gene expression studies on TSC tuber specimens focused either on selected cDNA sequences12, 13 or used mRNA14 or microRNA (miRNA) microarray hybrid- ization platforms15. Advances in high-throughput RNA sequencing (RNA-seq) technol- ogy coupled with sophisticated bioinformatics methods have provided a revolutionary means to systematically map transcriptional units of the human genome16, 17. Indeed, RNA-seq has led to a more profound appreciation of the intricate nature of both the coding and non-coding transcriptome of the human brain18, 19. Moreover, the non-coding units of the human transcriptome, particularly small RNA species that include miRNA, have emerged as important modifiers of the protein coding transcriptome and, in turn, disease phenotype19-22. A comprehensive, parallel scan of both the protein coding and small non-coding brain transcriptome of TSC patients by RNA-seq has never been per- formed. Thus, we here aimed to first map the protein coding and small non-coding RNA species in the brain transcriptome of TSC patients and second, to identify significantly altered cellular signaling pathways in TSC patients that may be modified by miRNA for a better delineation of the complex pathological signaling pathways and networks seen in TSC.
Methods
Human specimens
The cortical specimens from TSC and control patients included in this study were selected from the archives of the departments of neuropathology of the Academic Medical Center (AMC, University of Amsterdam, The Netherlands), the University Medical Center Utrecht (UMCU, The Netherlands), Motol University Hospital (Prague, Czech Republic) and Medical University Vienna (MUV, Austria). Informed consent was obtained for the use of brain tissue and for access to medical records for research pur- poses. We evaluated 12 TSC specimens (10 surgical specimens and 2 autopsy specimens) from whom we obtained anatomically well-preserved neocortical tubers tissue and suf- ficient clinical data. All patients fulfilled the diagnostic criteria for definite TSC23, 24. The surgical cases had pharmacologically intractable epilepsy (daily seizures) and underwent extensive pre-surgical evaluation6. Patient characteristics are summarized in Table 1. The following clinical data were extracted from medical records: TSC1/TSC2 mutation status, gender, localization of the resected area, age at seizure onset, duration of active epilepsy, antiepileptic drug (AED) management at time of surgery and presence/absence of intel-
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