Explore The Roles Of Gene Mutations In Malformations Of Cortical Development By Using High-throughput Sequencing

Epilepsy is a chronic neurologic disease, which is characterized by abnormal discharge synchronization of cortical neurons and followed by transitory central nervous system dysfunction. The annual incidence rate of epilepsy is about 7.0‰, and mostly affected the children and juveniles. Most of the patients after first-line antiepileptic drug therapy can achieve clinical ease, but about 30% of patients develop drug refractory epilepsy. According to the latest retrospective study, there is about 60% of patients who undergo epilepsy surgery are caused by malformations of cortical development, MCDs. Moreover, in intractable epilepsy patients less than 3 years old, MCD accounted for nearly 80%. Therefore, further molecular biology studies on the pathogenesis and epileptogenesis of MCD, will be a lot of help for the early diagnosis and individualized treatment of intractable epilepsy.There exists typical common pathological changes in MCD affected lesions, including destruction of coritcal lamellar structure and blur of the gray-white matter border. MCD consists of a wide spectrum of developmental disorders,such as tuberous sclerosis complex(TSC), focal cortical dysplasia (FCDs) and polymicrogyria et al. According to the classification criterion of FCDs established by the International League Against Epilepsy(ILAE) in 2011, FCDs can be preliminary divided into simple type and mixed type. Isolated FCDs can be further classified as FCD Ⅰ and FCD Ⅱ, which characterized by lamellar structure malformations and gray-white matter border abnormalities (FCD Ⅰ, FCD Ⅱ), and sporadic malformed cells including dysmorphic neurons (DNs) (FCD Ⅱa, FCD Ⅱb) and balloon cells (BCs) (FCD Ⅱb). TSC is a rare multisystem autosomal dominant disease that causes similar brain tissue pathological changes founded in FCD Ⅱb patients, including large amount of DNs and giant cells (GCs).In recent years, studies found that the epileptogenesis of FCDs is rather complex, which involved in aberrant ion channels, excessive inflammatory reaction, and abnormal extracellular matrix components et al. Most FCD Ⅱ occurs sporadically, and this implies somatic mutations in key genes associated with cell cycle regulation and neuronal migration as the underlying disorder mechanisms. Furthermore, exploring the downstream function of DNA mutations is more important. With the rise of the transcriptome high throughput technology, it is more convenient and effective to perform the functional genomics studies. It will deepen our understanding of the underlying the pathogenesis and epileptogenesis of FCD patients by using the WES and RNA-seq technology.For the current treatment of MCD related epilepsy, clinical antiepileptic drugs are most commonly focused on the selective ion channels targets, however there is less researches focused on the non-selective ion channels. Recently, more studies have focused on the non-selective TRP channels, which located mostly on the plasma membrane of a wide range of animal cells. These channels are grouped into 7 subtypes, such as TRPA, TRPV, TRPC,TRPM et al. TRP channels are non selectively permeable to cations via the activation of internal or external agonist. TRPC is a important subtype of TRP channel, which mainly distributed in animal brain tissues and mostly in the membrane of neurons, glial cells and endothelial cells. Previous study found that TRPC4 is involved in the epileptiform burst firing of hippocampus neurons. In addition, TRPC4 also participate in the aberrant excitatory/inhibitory neurotransmitter release process, which may disrupt the cortical"excitability/inhibition" balance, thus causing the epilepticus insultus. Therefore, we assumed that TRPC4 may also be involved in the epileptogenicity of malformations in cortical development-associated epilepsy.In this study, we make a comprehensive study of the exon region mutations and the downstream transcriptome information of FCD Ⅱ, through the usage of WES, RNA-seq and bioinformatics method. In addition, we analysed the mRNA and protein expression of TRPC4 in FCD Ⅱ and TSC cortical lesions and in control cortex (CTX) samples. Moreover, the specific cellular distribution of TRPC4 in the FCD Ⅱ and TSC samples were analysed using immunohistochemistry and immunofluorescence. The results are presented as following:Ⅰ . FCDs associated gene mutation analysis based on whole exome sequencing1. WES and bioinformatics analysis showed that the common and specific gene mutations of FCD Ⅱ are mainly distributed in GABAergic inhibition signaling, mTOR signaling, cell cycle regulation signaling and TRP channels;2. For the SNV mutations of FCD Ⅱ specimens analysis, we found that common mutations of FCD Ⅱ are involved in mTOR signaling and cell cycle G1 signaling, including mTOR, PTEN and CDC27 gene; the specific SNV variations of FCD Ⅱa are neuroblastoma breakpoint family (NBPF) associated gene, such as NBPF4, NBPF10, NBPF14 and NBPF15;the specific SNV mutations of FCD Ⅱb are mostly GABAergic inhibition gene, including SLC6A11, SLC12A5 mutations. Upon the mutations are strongly associated with CNS development and epileptogenesis, we assume that these gene mutations are potential major genes underlying the pathogenesis and epileptogenesis of FCD Ⅱ;3. For the copy number variations (CNVs) analysis of FCD Ⅱ patients, specific axon guidance process genes EFNB2, EFNA1, PLX-NA1 and NTNG2 are presented in FCD Ila specimen. In addition, TSC1、TSC2 and CDC27 gene copy number decreases are specific showed in FCD Ⅱb patients. Moreover, increased gene copy number of TRPV1, TRPV4 and TRPC4 are commonly existed in FCDⅡ. The CNV of FCDⅡ are mainly associated cell proliferation, differentiation and neuronal electrical activity et al, which indicated that these gene expression changes may involve in the disease development of FCD Ⅱ.Ⅱ. FCDs associated transcriptome analysis based on RNA-seq1. RNA-seq and bioinformatics analysis showed that the data are consistent with previous studies. There are about 464 common differentially expressed genes in FCD Ⅱ, 536 specific differentially expressed genes in FCD Ⅱa and 687 specific differentially expressed genes in FCD Ⅱb;2. Firstly, function clustering analysis was conducted to investigate the FCD Ⅱ common differentially expressions, we found that the significantly changed genes are VEGFA、KCNJ10、CACNG4、TRPV1、TRPC4、P2X7R and P2Y12R, which are clustered into the angiogenesis, cation transportation, and P2 receptor signaling. Based on the review of related papers, the genes above may be involved in the epileptogenesis of FCD Ⅱ, and may become new targets of specific treatment of intractable epilepsy;3. Then, we conducted function clustering analysis of FCD Ⅱa and FCD Ⅱb specific differentially expressed genes. The FCD Ⅱa specific genes are mainly ion channel associated genes, including upregulated SLC13A5、SLC1A6、GR1N3A、NMUR2 and KCNK5 genes; For FCD Ⅱb specimen, we found that the downregulated genes are CDKN1C、CDKN2A and GDNF, which focused on the cell cycle regulation and central nervous system development signaling, these specific gene expression may shed light on the pathogenesis of FCD II subtype.Ⅲ. Expression and distribution of TRPC4 and PLCD1 in MCD specimens1. RT-PCR and Western Blot data have shown the expression of TRPC4 mRNA and protein in FCD Ⅱ, TSC and CTX brain tissues. Further immunohistochemistry experiments indicated that there is a weak to moderate expression of TRPC4 in neurons, glial cells and endothelial cells.2. Compared with the control cortex, there was an obviously increasing of TRPC4 mRNA and protein expression in MCD cortical homogenate (p < 0.01), while no significant differences between FCD Ⅱ and TSC were shown (p > 0.05). Single-labelling immunohistochemistry and double-labelling immunofluorescence showed that moderate to strong TRPC4 staining signal was detected in DNs, BCs, GCs, glial cells and endothelial cells of FCD Ⅱ and TSC, which there are significant difference between MCDs and CTX (p <0.01), however no significant differences between FCD Ⅱ and TSC specimens (p > 0.05).The double-labelling experiments showed that most NeuN-labelled DNs, BCs, GCs were TRPC4-positive, and TRPC4 and GFAP were co-expressed in most astrocytes and some BCs,GCs in the FCD Ⅱ and TSC samples. In addition, colocalization of TRPC4 with vGLUT1,GABA and GAD65/67 was observed in DNs, BCs and GCs of MCD specimens. Therefore,we hypothesized that overexpression of TRPC4 may play an important role in the epileptogenesis of these cortical malformations.3. Furthermore, the upstream interaction factor of TRPC4, PLCD1, is significantly increased in FCD Ⅱ and TSC samples compared with the CTX samples (p < 0.01). which mainly expressed in malformed cells, glial cells and endothelial cells. Upon the results, we speculated that PLCD1 may be involved in epileptogenesis in FCD Ⅱand TSC patients by modulating the activation of TRPC4.Ⅳ. In summary, our study reveal that:1. In cortical lesions of FCD Ⅱ, there are many potential pathogenic gene mutations with the distribution of mTOR signaling, cell cycle regulation signaling (mTOR、PTEN、TSC and CDC27) which may participate in the pathogenesis of FCD Ⅱ, GABAergic inhibition signaling and TRP channels (SLC6A11K SLC12A5 and TPRC4) which may contribute to the high genetic susceptibility to epilepsy;2. We conduct the RNA-seq experiment to comprehensively analyse the transcriptome information of FCD II for the first time. After bioinformatics analysis, we found that cell cycle regulation and central nervous system development signaling genes (CDKN1C and CDKN2A) are associated with the pathogenesis of ballon cells in FCD Ⅱ. Meanwhile,abnormal expression of ion channel associated genes SLC13A5、SLC1A6、KCNK5 and TRPC4 may disturb the balance between excitatory and inhibitory neural circuits in the human cerebral cortex, thus enhancing the seizure activity in FCD II lesions.3. Furthermore, TRPC4 and its upstream interaction factor PLCD1 are up regulated in MCD specimens, and mainly located in malformed cells and endothelial cells.In conclusion, mTOR signaling pathway, cell cycle regulation and ion channels associated genes may play extremely important roles in pathogenesis and epileptogenesis of MCD disease,meanwhile,TRPC4 may be a new target for MCD associated epilepsy treatment.