Identification of a Novel Deletion Mutation (c.1780delG) and a Novel Splice-Site Mutation (c.1412-1G&gt;A) in the <Emphasis Type="Italic">CCM1/KRIT1</Emphasis> Gene Associated with Familial Cerebral Cavernous Malformation in the Chinese Population

Cerebral cavernous malformation (CCM) is a congenital vascular anomaly predominantly located within the central nervous system. Its familial forms (familial cerebral cavernous malformation (FCCM)), inherited in an autosomal dominant manner with incomplete penetrance, are attributed to mutations in CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10 genes. To date, little is known about the genetic alterations leading to FCCM in the Chinese population. We aimed to investigate the genetic defect of FCCM by DNA sequencing in Chinese families. This study enrolled five Chinese families with FCCM. All index cases underwent surgical treatment and were diagnosed with CCM by pathology; their relatives were diagnosed based on radiological and/or pathological evidence. Genomic DNA was extracted from peripheral blood and amplified using polymerase chain reaction (PCR) for DNA sequencing. The five families comprised a total of 21 affected individuals: 12 of these were symptomatic, and 9 were asymptomatic. Sequence analyses in the index patients disclosed three heterozygous loss-of-function mutations in the CCM1/KRIT1 gene in three families, respectively: a novel deletion mutation (c.1780delG; p.Ala594HisfsX67) in exon 16, a novel splice-site mutation (c.1412-1G>A) in the splice acceptor site in intron 13, and a previously described 4-bp deletion (c.1197_1200delCAAA; p.Gln401ThrfsX10) in exon 12. All of these mutations are predicted to cause a premature termination codon to generate a truncated Krev interaction trapped 1 (Krit1) protein. These mutations segregated in affected relatives. Our findings provided new CCM1 gene mutation profiles, which help to elucidate the pathogenesis of FCCM and will be of great significance in genetic counseling.     

<Emphasis Type="Italic">Dicer1</Emphasis> Ablation Impairs Responsiveness of Cerebellar Granule Neuron Precursors to Sonic Hedgehog and Disrupts Expression of Distinct Cell Cycle Regulator Genes

Granule neuron precursors (GNPs) proliferate under the influence of Sonic hedgehog (Shh) that is secreted by Purkinje neurons during early postnatal cerebellar development. To investigate microRNA (miRNA) function in this developmental process, we conditionally deleted the Dicer1 gene under the activity of human glial fibrillary acidic protein (hGFAP) promoter. We report that Dicer1-ablated GNPs display decreased proliferation and survival at early postnatal stages and that the proliferation defect of mutant GNPs cannot be rescued by treatment of an Shh agonist in vitro as assayed by 5-bromo-2′-deoxyuridine (BrdU) pulse labeling and Shh target gene expression detection. Further analysis reveals that the expression of distinct cell cycle regulator genes including cell cycle inhibitor, CDKN1a (p21), selectively increases in Dicer1-ablated GNPs. Subsequently, we demonstrate that miR-17-5p exhibits high expression level in the developing cerebellum and that transfection of a synthetic miR-17-5p mimic downregulates p21 protein expression in GNPs and promotes proliferation of GNPs in culture. Therefore, Dicer1 ablation impairs Shh-induced GNP proliferation by disrupting the expression of distinct cell cycle regulator genes that are targets of miR-17～92 cluster members. This study establishes a molecular link between miRNAs and cell cycle progression in the proliferating GNPs during normal cerebellar development and may facilitate miRNA application in treating medulloblastoma.     

A Novel <Emphasis Type="Italic">KRIT1/CCM1</Emphasis> Gene Insertion Mutation Associated with Cerebral Cavernous Malformations in a Chinese Family

Familial cerebral cavernous malformation (FCCM) is a vascular malformation disorder that closely associated with three identified genes: KRIT1/CCM1, MGC4607/CCM2, and PDCD10/CCM3. Here, we present a Chinese family affected by FCCM due to a novel KRIT1/CCM1 insertion mutation. The proband was hospitalized for sudden unconsciousness and underwent surgical treatment. The section of lesions showed classical cavernous-dilated vessels without intervening brain parenchyma, and hemosiderin-laden macrophages were accumulated in the surrounding tissue. In addition, magnetic resonance imaging (MRI) showed severe multiple cerebral cavernous malformation (CCM) lesions in cerebrum, brainstem, and cerebellum in other affected subjects. Especially, for the proband’s mother, hundreds of lesions were presented, and a few lesions were found in the expanded lateral ventricle (Evans’ index =0.33). Moreover, she showed the similar symptoms of hydrocephalus, including headache, dizziness, and diplopia. It was extremely rare in previous reports. To date, the genetic alterations leading to FCCM in Chinese population remain largely unknown. We investigated genetic defects of this family. Sequence analyses disclosed a novel heterozygous insertion mutation (c.1896_1897insT; p.Pro633SerfsTer22) in KRIT1/CCM1. Moreover, our real-time PCR results revealed that the mRNA level of KRIT1/CCM1 were significantly decreased in FCCM subjects (CCM family =0.42 ± 0.20 vs. healthy control =1.01 ± 0.16, P = 0.004). It indicated that this mutation could cause KRIT1/CCM1 functional mRNA deficiency. It may be closely related with the pathogenesis of FCCM. Our findings provided a new gene mutation profile which will be of great significance in early diagnosis and appropriate clinical surveillance of FCCM patients.     

Circulating miR-126 and miR-130a levels correlate with lower disease risk,disease severity,and reduced inflammatory cytokine levels in acute ischemic stroke patients

To investigate the correlations of five angiogenesis-related miRNA (miR-126, miR-130a, miR-222, miR-218, and miR-185) expression levels with risk, severity, and inflammatory cytokines levels in acute ischemic stroke (AIS) patients. A total of 148 AIS patients and 148 age- and gender-matched controls were consecutively enrolled. Blood samples were collected from AIS patients and controls, and plasma was separated for miRNAs and cytokine level detection. Plasma levels of miRNAs were evaluated by real-time qPCR method, and inflammatory cytokine levels were detected using an enzyme-linked immunosorbent assay (ELISA). Plasma miR-126 and miR-130a expression levels in AIS patients were lower than those of controls, while the levels of miR-222, miR-218, and miR-185 were elevated in AIS patients compared with controls. After pooling the five miRNA expression levels together, the area under the curve (AUC) for predicting AIS risk was 0.840 (95% CI 0.795–0.885) with a sensitivity of 83.8% and a specificity of 69.6% at the best cut-off point. Plasma miR-126 (r?=???0.402, P?<?0.001) and miR-130a (r?=???0.161, P?=?0.050) levels were negatively correlated with NIHSS scores, while plasma miR-218 level was positively correlated with NIHSS scores (r?=?0.471, P?<?0.001). Most importantly, plasma miR-126 expression was negatively correlated with TNF-α (r?=???0.168, P?=?0.041), IL-1β (r?=???0.246, P?=?0.003), and IL-6 (r?=???0.147, P?=?0.035) levels, while miR-130a expression was negatively correlated with TNF-α (r?=???0.287, P?<?0.001), IL-1β (r?=???0.168, P?=?0.041), and IL-6 (r?=???0.239, P?=?0.003) expression levels and positively associated with IL-10 level (r?=?0.261, P?=?0.001). Circulating miR-126 and miR-130a levels correlate with lower disease risk, decreased disease severity, and reduced inflammatory cytokine levels in AIS patients.     

A Novel <Emphasis Type="Italic">CCM1</Emphasis>/<Emphasis Type="Italic">KRIT1</Emphasis> Heterozygous Nonsense Mutation (c.1864C&gt;T) Associated with Familial Cerebral Cavernous Malformation: a Genetic Insight from an 8-Year Continuous Observational Study

Cerebral cavernous malformation (CCM) is a congenital vascular abnormality that predominantly affects the central nervous system, but that sometimes encroaches other vital tissues, including the retina, skin, and even liver. The familial form of CCM (FCCM) is considered to be an autosomal dominant disease with incomplete penetrance and variable expression, which is often attributed to mutations in three genes: CCM1, CCM2, and CCM3. We screened a Chinese family diagnosed with FCCM by using Sanger sequencing. A 29-year-old male proband with cutaneous angiomas was pathologically diagnosed but presented with an atypical form of CCM as revealed by magnetic resonance imaging (MRI) findings, prompting further clinical evaluation and genetic analyses of him and his immediate family. We performed continuous observation over an 8-year period using MRI gradient echo imaging and susceptibility-weighted imaging of these individuals. Sanger sequencing of the CCM1, CCM2, and CCM3 genes identified a novel heterozygous nonsense nucleotide transition (c.1864C>T; p.Gln622X) in exon 17 of the CCM1/KRIT1 gene; this mutation was predicted to cause a premature stop codon (TAG) at nucleotides 1864 to 1866 to generate a truncated Krev interaction trapped 1 (Krit1) protein of 621 amino acids. During this long-term observational study, one of the enrolled family members with neurological deficits progressed to a stage indicative of brain surgery. This study provides a new CCM gene mutation profile, which highlights the significance of genetic counseling for individuals suspected of having this condition.     

First large genomic inversion in familial cerebral cavernous malformation identified by whole genome sequencing

Familial cerebral cavernous malformations (CCMs) predispose to seizures and hemorrhagic stroke. Molecular genetic analyses of CCM1, CCM2, and CCM3 result in a mutation detection rate of up to 98%. However, only whole genome sequencing (WGS) in combination with the Manta algorithm for analyses of structural variants revealed a heterozygous 24 kB inversion including exon 1 of CCM2 in a 12-year-old boy with familial CCMs. Its breakpoints were fine-mapped, and quantitative analysis on RNA confirmed reduced CCM2 expression. Our data expand the spectrum of CCM mutations and indicate that the existence of a fourth CCM disease gene is rather unlikely.     

A direct regulatory link between microRNA-137 and <Emphasis Type="Italic">SHANK2</Emphasis>: implications for neuropsychiatric disorders

Background

Mutations in the SHANK genes, which encode postsynaptic scaffolding proteins, have been linked to a spectrum of neurodevelopmental disorders. The SHANK genes and the schizophrenia-associated microRNA-137 show convergence on several levels, as they are both expressed at the synapse, influence neuronal development, and have a strong link to neurodevelopmental and neuropsychiatric disorders like intellectual disability, autism, and schizophrenia. This compiled evidence raised the question if the SHANKs might be targets of miR-137.

Methods

In silico analysis revealed a putative binding site for microRNA-137 (miR-137) in the SHANK2 3′UTR, while this was not the case for SHANK1 and SHANK3. Luciferase reporter assays were performed by overexpressing wild type and mutated SHANK2-3′UTR and miR-137 in human neuroblastoma cells and mouse primary hippocampal neurons. miR-137 was also overexpressed or inhibited in hippocampal neurons, and Shank2 expression was analyzed by quantitative real-time PCR and Western blot. Additionally, expression levels of experimentally validated miR-137 target genes were analyzed in the dorsolateral prefrontal cortex (DLPFC) of schizophrenia and control individuals using the RNA-Seq data from the CommonMind Consortium.

Results

miR-137 directly targets the 3′UTR of SHANK2 in a site-specific manner. Overexpression of miR-137 in mouse primary hippocampal neurons significantly lowered endogenous Shank2 protein levels without detectable influence on mRNA levels. Conversely, miR-137 inhibition increased Shank2 protein expression, indicating that miR-137 regulates SHANK2 expression by repressing protein translation rather than inducing mRNA degradation.To find out if the miR-137 signaling network is altered in schizophrenia, we compared miR-137 precursor and miR-137 target gene expression in the DLPFC of schizophrenia and control individuals using the CommonMind Consortium RNA sequencing data. Differential expression of 23% (16/69) of known miR-137 target genes was detected in the DLPFC of schizophrenia individuals compared with controls. We propose that in further targets (e.g., SHANK2, as described in this paper) which are not regulated on RNA level, effects may only be detectable on protein level.

Conclusion

Our study provides evidence that a direct regulatory link exists between miR-137 and SHANK2 and supports the finding that miR-137 signaling might be altered in schizophrenia.

     

Common genetic variation within <Emphasis Type="Italic">miR-146a</Emphasis> predicts disease onset and relapse in multiple sclerosis

Despite extensive studies focusing on the changes in expression of microRNAs (miRNAs) in multiple sclerosis (MS) compared to healthy controls, few studies have evaluated the association of genetic variants of miRNAs with MS clinical course. We investigated whether a functional polymorphism in the MS associated miR-146a gene predicted clinical course (hazard of conversion to MS and of relapse, and annualized change in disability), using a longitudinal cohort study of persons with a first demyelinating event followed up to their 5-year review. We found the genotype (GC+CC) of rs2910164 predicted relapse compared with the GG genotype (HR=2.09 (95% CI 1.42, 3.06), p=0.0001), as well as a near-significant (p=0.07) association with MS conversion risk. Moreover, we found a significant additive interaction between rs2910164 and baseline anti-EBNA-1 IgG titers predicting risk of conversion to MS (relative excess risk due to interaction [RERI] 2.39, p=0.00002) and of relapse (RERI 1.20, p=0.006). Supporting these results, similar results were seen for the other EBV-correlated variables: anti-EBNA-2 IgG titers and past history of infectious mononucleosis. There was no association of rs2910164 genotype for disability progression. Our findings provide evidence for miR-146a and EBV infection in modulating MS clinical course.     

Evaluation of Selected MicroRNAs Expression in Remission Phase of Multiple Sclerosis and Their Potential Link to Cognition,Depression, and Disability

Accumulating data suggests that miRNAs might play a major role in neuroinflammatory processes. Therefore, our study aimed to first estimate the levels of miR-155, miR-326, and miR-301a in serum of RR-MS patients in the remission phase and then compare the levels of the examined miRNAs at different times after relapse. In this study, 36 RR-MS patients in the remission phase took part. We analyzed two subgroups of RR-MS: one, 1 to 2 months after completing steroid treatment during relapse (post-acute; n = 13) and the other, over 2 years without any relapse (stable; n = 23). Moreover, we made correlations between these biochemical results and clinical parameters of cognitive impairment, depression, and disability. The obtained results presented downregulation of miR-155 and miR-301a (in 94% and 51% samples, respectively) and overexpression of miR-326 (in 72% samples) in RR-MS patients. Moreover, we observed a positive correlation between the relative expression of miRNAs and BDI (Beck Depression Index) for miR-326 (rho = 0.385459, p = 0.022210; Spearman’s rank correlation) and miR-301a (rho = 0.435131, p = 0.008991; Spearman rank correlation). We also observed the differences in expression levels between the post-acute and stable phases of RR-MS. The expression levels of miR-301a and miR155 were higher in the post-acute vs. stable phase of remission (2.385 vs. 0.524 and 0.594 vs. 0.147; respectively). Our study, for the first time, presents miRNA expression differences in two stages of remission: post-acute and stable.     

microRNA profiling: increased expression of <Emphasis Type="Italic">miR-147a</Emphasis> and <Emphasis Type="Italic">miR-518e</Emphasis> in progressive supranuclear palsy (PSP)

Progressive supranuclear palsy is a sporadic neurodegenerative disorder. Genetic, environmental, and possibly epigenetic factors contribute to disease. In order to better understand the potential role of epigenetic changes in progressive supranuclear palsy, we investigated whether some microRNAs and their target genes are dysregulated. We analyzed expression of 372 well-characterized microRNAs in forebrains of a total of 40 patients and of 40 controls using TaqMan arrays and SYBR Green quantitative real-time PCR. The exploratory cohort included forebrains from 20 patients and 20 controls provided by the Erasmus Medical Centre in Rotterdam, Netherlands. Confirmatory samples were from Jacksonville, Florida, and from Melbourne, Australia. Both microRNA profiling and SYBR Green quantitative real-time PCR revealed significant upregulation of miR-147 (miR-147a) and miR-518e in the exploratory cohort. Highly increased expression of these two microRNAs was validated in the confirmatory samples. Target genes of miR-147a (NF1, ACLY, ALG12) and of miR-518e (CPEB1, JAZF1, RAP1B) were repressed in patients’ forebrains. The results suggest that dysregulation of specific microRNAs contributes to disease by repressing target genes involved in various cellular functions.     

Identification of Critical Genes and miRNAs Associated with the Development of Parkinson’s Disease

The purpose of this study was to explore the key mechanism involved in the pathogenesis of Parkinson’s disease (PD) based on microarray analysis. The expression profile data of GSE7621, which contained 9 substantia nigra tissues isolated from normals and 16 substantia nigra tissues isolated from PD patients, was obtained from Gene Expression Omnibus. The differentially expressed genes (DEGs) were screened, followed by functional enrichment analysis and protein-protein interaction (PPI) network construction. After the miRNAs regulating the DEGs were predicted, the miRNA-DEG regulatory network was then constructed. Besides, the 6-hydroxydopamine rat model of PD was established and the expression of key DEGs and miRNA was detected. A total of 388 DEGs were identified, including 218 upregulated genes and 170 downregulated ones. Tyrosine hydroxylase (TH) and solute carrier family 6 member 3 (SLC6A3) were significantly related to the functional terms of catecholamine biosynthetic process and dopamine biosynthetic process. TH and SLC6A3 were hub nodes in the PPI network. EBF3 could be targeted by miR-218. Moreover, TH and SLC6A3 were found downregulated in the 6-OHDA rat model of PD, while miR-218 was markedly upregulated. Our results reveal that SLC6A3, TH, and EBF3 targeted by miR-218 could be involved in PD. These molecules might provide a new insight into the development of therapeutic strategies for PD.     

A newly distal hereditary motor neuropathy caused by a rare <Emphasis Type="Italic">AIFM1</Emphasis> mutation

In two siblings, who suffer from an early childhood-onset axonal polyneuropathy with exclusive involvement of motor fibers, the c.629T>C (p.F210S) mutation was identified in the X-linked AIFM1 gene, which encodes for the apoptosis-inducing factor (AIF). The mutation was predicted as deleterious, according to in silico analysis. A decreased expression of the AIF protein, altered cellular morphology, and a fragmented mitochondrial network were observed in the proband’s fibroblasts. This new form of motor neuropathy expands the phenotypic spectrum of AIFM1 mutations and therefore, the AIFM1 gene should be considered in the diagnosis of hereditary motor neuropathies.     

IL-17 Exerts Anti-Apoptotic Effect via miR-155-5p Downregulation in Experimental Autoimmune Encephalomyelitis

Multiple sclerosis is an autoimmune, neurodegenerative disease, affecting mostly young adults and resulting in progressive disability. It is a multifactorial disorder, with important involvement of both cellular and epigenetic components. Among the epigenetic factors, microRNAs are currently intensively investigated in the context of multiple sclerosis. It has been shown that their biogenesis and function may be regulated by various cytokines. IL-17, a hallmark cytokine of Th17 cells, has been thought to function predominantly as a pro-inflammatory factor, leading to increased disease symptoms. However, there are several studies indicating its protective role during inflammatory process. In this work, we have assessed the impact of high-dose IL-17 administration on microRNAs’ expression profile during the preclinical stage of EAE. For selected microRNA, we have performed computational analysis of its potential target mRNAs and cellular pathways. Based on results obtained from in silico analysis, we have chosen genes from neurotrophin signaling pathway for further experiments—BDNF, HRAS, and BCL2. Results obtained in this study suggested that high dose of IL-17 exerts protective activity via miR-155-5p downregulation. Increased expression of all studied genes, especially BCL2, indicated a potential anti-apoptotic function of IL-17 during the preclinical phase of EAE.