Mapping cell diversity in human sporadic cerebral cavernous malformations.

BACKGROUND: Cerebral cavernous malformation (CCM) is a low-flow, bleeding-prone vascular disease that can cause cerebral hemorrhage, seizure and neurological deficits. Its inheritance mode includes sporadic or autosomal dominant inheritance with incomplete penetrance, namely sporadic CCM (SCCM) and familial CCM. SCCM is featured by single lesion and single affection in a family. Among CCM patients especially SCCM, the pathogenesis of the corresponding phenotypes and pathological features or candidate genes have not been fully elucidated yet. METHODS: Here, we performed in-depth single-cell RNA sequencing (scRNA-Seq) and bulk assay for transposase-accessible chromatin sequencing (ATAC-Seq) in SCCM and control patients. Further validation was conducted for the gene of interest using qPCR and RNA in situ hybridization (RNA FISH) techniques to provide further atlas and evidence for SCCM generative process. RESULTS: We identified six cell types in the SCCM and control vessels and found that the expression of NEK1, RNPC3, FBRSL1, IQGAP2, MCUB, AP3B1, ESCO1, MYO9B and PVT1 were up-regulated in SCCM tissues. Among the six cell types, we found that compared with control conditions, PVT1 showed a rising peak which followed the pseudo-time axis in endothelial cell clusters of SCCM samples, while showed an increasing trend in smooth muscle cell clusters of SCCM samples. Further experiments indicated that, compared with the control vessels, PVT1 exhibited significantly elevated expression in SCCM samples. CONCLUSION: In SCCM conditions, We found that in the process of development from control to lesion conditions, PVT1 showed a rising peak in endothelial cells and showed an increasing trend in smooth muscle cells at the same time. Overall, there was a significantly elevated expression of NEK1, RNPC3, FBRSL1, IQGAP2, MCUB, AP3B1, ESCO1, MYO9B and PVT1 in SCCM specimens compared to control samples.

Corrigendum: Alterations of the Gut Microbiota in Multiple System Atrophy Patients.

[This corrects the article DOI: 10.3389/fnins.2019.01102.].

Alterations of the Gut Microbiota in Multiple System Atrophy Patients.

Multiple system atrophy (MSA) is a fatal neurodegenerative disease, and the pathogenesis is still quite challenging. Emerging evidence has shown that the brain-gut-microbiota axis served a pivotal role in neurological diseases; however, researches utilizing metagenomic sequencing to analyze the alteration in gut microbiota of MSA patients were quite rare. Here, we carried out metagenomic sequencing in feces of 15 MSA patients and 15 healthy controls, to characterize the alterations in gut microbial composition and function of MSA patients in mainland China. The results showed that gut microbial community of MSA patients was significantly different from healthy controls, characterized by increased genus Akkermansia and species Roseburia hominis, Akkermansia muciniphila, Alistipes onderdonkii, Streptococcus parasanguinis, and Staphylococcus xylosus, while decreased genera Megamonas, Bifidobacterium, Blautia, and Aggregatibacter and species Bacteroides coprocola, Megamonas funiformis, Bifidobacterium pseudocatenulatum, Clostridium nexile, Bacteroides plebeius, and Granulicatella adiacens. Further, functional analysis based on the KEGG database revealed aberrant functional pathways in fecal microbiome of MSA patients. In conclusion, our findings provided evidence for dysbiosis in gut microbiota of Chinese MSA cohorts and helped develop new testable hypotheses on pathophysiology of MSA.

RNA Expression Profile and Potential Biomarkers in Patients With Spinocerebellar Ataxia Type 3 From Mainland China.

Long non-coding RNAs (lncRNAs) play an important role in growth, development, and reproduction and undoubtedly contribute to the pathogenesis and progression of diseases. Emerging evidence suggests the involvement of lncRNAs as regulatory factors in pathological conditions, including some neurodegenerative diseases. Spinocerebellar Ataxia Type 3/Machado-Joseph Disease (SCA3/MJD) has a prominent prevalence in China. Because the role of lncRNAs in SCA3/MJD pathogenesis has not yet been investigated, we conducted a pilot study to investigate the expression profile of lncRNAs by high-throughput sequencing in 12 patients and 12 healthy individuals. The sequencing analysis detected 5,540 known and 2,759 novel lncRNAs. Six lncRNAs were confirmed to be differentially expressed in peripheral blood mononuclear cells between SCA3/MJD patients and healthy individuals and were further validated in cerebellar tissue. Based on these results, NONHSAT022144.2 and NONHSAT165686.1 may be involved in the pathogenesis of SCA3/MJD and may be potential biomarkers for SCA3/MJD. Together with NONHSAT022144.2 and NONHSAT165686.1, the other four novel lncRNAs increase our understanding of lncRNA expression profile.

Identification of a potential exosomal biomarker in spinocerebellar ataxia Type 3/Machado-Joseph disease.

Aim: To identify spinocerebellar ataxia Type 3 (SCA3)-related exosomal biomarkers and the underlying mechanisms. Materials & methods: Exosomal RNAs from plasma and cerebrospinal fluid (CSF) were extracted from 24 SCA3 patients and 22 controls, respectively. Small RNA sequencing and quantitative PCR verification were performed. Gene ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway analyses of the results were carried out. Results: One novel miRNA is notably downregulated in plasma-derived exosomes, while upregulated in CSF-derived exosomes of SCA3 patients. Besides, it is successively upregulated in CSF-derived exosomes from Type 1, Type 2 and Type 3 groups. The downstream target genes were enriched in protein processing in endoplasmic reticulum and axon guidance. Conclusion: One exosomal biomarker was identified in SCA3, and this is the first time to report an exosomal miRNA as a biomarker in SCA3 internationally.

Polymorphisms in DNA methylation-related genes are linked to the phenotype of Machado-Joseph disease.

DNA methylation has been reported as an important regulator of genomic structure stability, including large tandem repeats. To test the modulation effect of variants in DNA methylation-related genes on distribution of expanded (CAG)n alleles and age at onset (AO) of patients with Machado-Joseph disease (MJD), we conducted an association analysis on 23 selected SNPs in these genes in 613 patients with MJD and 581 controls. There were significant differences in the distribution of rs12957023 between patients and controls (OR = 1.296, p = 0.007 and OR = 1.206, p = 0.008, for genotype and alleles, respectively). The distribution of (CAG)n size was also different between patients carrying a CC and the other genotypes (TT and TC, p = 0.011 for expanded (CAG)n and p = 0.012 for normal size alleles), indicating that DNA methylation might modulate the (CAG)n instability. We found also that rs13420827 in DNMT3A and rs7354779 in DNMT3L contribute to AO of MJD (p = 0.019 and p = 0.008, respectively). In conclusion, our data provide the first evidence that SNPs in DNA methylation-related genes may contribute to (CAG)n instability and modulate the AO of this disease.

Cerebellar lncRNA Expression Profile Analysis of SCA3/MJD Mice.

Spinocerebellar ataxia type 3 (SCA3) or Machado-Joseph disease (MJD) is the most common autosomal dominant spinocerebellar ataxia in China with highly clinical heterogeneity, such as progressive cerebellar ataxia, dysarthria, pyramidal signs, external ophthalmoplegia, dysphagia, and distal muscle atrophy. It is caused by the abnormal expansion of CAG repeats in a coding region of ATXN3. However, by focusing on the ATXN3 itself cannot fully explain the heterogeneous clinical features of SCA3/MJD. With the discovery of the increasing number of long noncoding RNAs (lncRNAs) that are believed to be involved in spinocerebellar ataxia type 8 (SCA8) and Huntington disease (HD), we wonder whether the lncRNAs are differentially expressed in the SCA3/MJD patients compared to the nonpatients. As the first step, we used lncRNA-Seq to investigate differential expression of the lncRNAs in the SCA3/MJD mice. Two known lncRNAs, n297609 and n297477, and a novel lncRNA TCONS_00072962 have been identified in SCA3/MJD mice with abnormal expression. The first discovery of the novel lncRNA TCONS_00072962 enriched the lncRNA expression profile in the SCA3/MJD mouse model.

Investigation on modulation of DNA repair pathways in Chinese MJD patients.

It has been reported that DNA repair pathways could modify age at onset (AO) in Huntington disease (HD) and spinocerebellar ataxias. We genotyped 22 SNPs from DNA repair pathways in a large cohort of 798 Chinese Machado-Joseph disease patients to investigate the association with AO, and no significant finding was observed. Our findings did not provide a strong evidence for the modulatory effect of DNA repair pathways on the AO of Chinese Machado-Joseph disease patients. Further analyses with more representative DNA repair-related SNPs in different populations are needed to identify new potential genetic modifiers.

Birt-Hogg-Dubé syndrome in two Chinese families with mutations in the FLCN gene.

BACKGROUND: Birt-Hogg-Dubé syndrome is an autosomal dominant hereditary condition caused by mutations in the folliculin-encoding gene FLCN (NM_144997). It is associated with skin lesions such as fibrofolliculoma, acrochordon and trichodiscoma; pulmonary lesions including spontaneous pneumothorax and pulmonary cysts and renal cancer. METHODS: Genomic DNA was extracted from peripheral venous blood samples of the propositi and their family members. Genetic analysis was performed by whole exome sequencing and Sanger sequencing aiming at corresponding exons in FLCN gene to explore the genetic mutations of these two families. RESULTS: In this study, we performed genetic analysis by whole exome sequencing and Sanger sequencing aiming at corresponding exons in FLCN gene to explore the genetic mutations in two Chinese families. Patients from family 1 mostly suffered from pneumothorax and pulmonary cysts, several of whom also mentioned skin lesions or kidney lesions. While in family 2, only thoracic lesions were found in the patients, without any other clinical manifestations. Two FLCN mutations have been identified: One is an insertion mutation (c.1579_1580insA/p.R527Xfs on exon 14) previously reported in three Asian families (one mainland family and two Taiwanese families); while the other is a firstly reviewed mutation in Asian population (c.649C > T / p.Gln217X on exon 7) that ever been detected in a French family. CONCLUSIONS: Overall, The detection of these two mutations expands the spectrum of FLCN mutations and will provide insight into genetic diagnosis and counseling of Birt-Hogg-Dubé syndrome.

Identifying SYNE1 Ataxia With Novel Mutations in a Chinese Population.

Objective: Variants in SYNE1 have been widely reported in ataxia patients in Europe, with highly variable clinical phenotype. Until now, no mutation of SYNE1 ataxia has been reported among the Chinese population. Our aim was to screen for SYNE1 ataxia patients in China and extend the clinicogenetic spectrum. Methods: Variants in SYNE1 were detected by high-throughput sequencing on a cohort of 126 unrelated index patients with unexplained autosomal recessive or sporadic ataxia. Pathogenicity assessments of SYNE1 variants were interpreted according to the ACMG guidelines. Potential pathogenic variants were confirmed by Sanger sequencing. Clinical assessments were conducted by two experienced neurologists. Results: Two Chinese families with variable ataxia syndrome were identified (accounting for 1.6%; 2/126), separately caused by the novel homozygous SYNE1 mutation (NM_033071.3: c.21568C>T, p.Arg7190Ter), and compound heterozygous SYNE1 mutation (NM_033071.3: c.18684G>A, p.Trp6228Ter; c.17944C>T, p.Arg5982Ter), characterized by motor neuron impairment, mental retardation and arthrogryposis. Conclusions: SYNE1 ataxia exists in the Chinese population, as a rare form of autosomal recessive ataxia, with a complex phenotype. Our findings expanded the ethnic, phenotypic and genetic diversity of SYNE1 ataxia.

Is the High Frequency of Machado-Joseph Disease in China Due to New Mutational Origins?

Machado-Joseph disease (MJD, also known as spinocerebellar ataxia 3 or SCA3) is the most common dominant ataxia worldwide, with an overall average prevalence of 1-5/100,000. To this date, two major ancestral lineages have been found throughout the world. In China, the relative frequency of MJD among the SCAs reaches as high as 63%, however, little is known about its mutational origin in this country. We analyzed 50 families with MJD patients in two or more generations to study the hypothesis that new mutational events have occurred in this population. Haplotypes based on 20 SNPs have shown new genetic backgrounds segregating with MJD mutations in our cohort from China. We found the "Joseph-derived" lineage (Joseph lineage with a G variant in rs56268847) to be very common among Chinese MJD patients. Moreover, we estimated the time for the origin of this MJD SNP background based on STR diversity flanking the (CAG)n of ATXN3. It was surprising to find that the Chinese MJD population originated from 8,000 to 17,000 years ago, far earlier than the previous literature reports, which will be an important evidence to explain the origin, spread and founder effects of MJD.

Let-7f Regulates the Hypoxic Response in Cerebral Ischemia by Targeting NDRG3.

microRNAs are a class of non-coding RNAs including approximately 22 nucleotides in length and play a pivotal role in post-transcriptional gene regulation. Currently, the role of miRNAs in the pathophysiology of ischemic stroke has been the subject of recent investigations. In particular, antagomirs to microRNA (miRNA) let-7f have been found to be neuroprotective in vivo, although the detailed function of let-7f during cerebral ischemia has not been fully illustrated. NDRG3 is an N-myc downstream-regulated gene (NDRG) family member that has been observed in the nuclei in most brain cells. Recently, a NDRG3-mediated lactate signaling, in which stabilized NDRG3 protein can promote angiogenesis and cell growth by activating the Raf-ERK pathway in hypoxia was discovered. In this study, we preliminarily explored the change in the expression of the NDRG3 protein which indicated that NDRG3 protein is an oxygen-regulated protein in neurons in rat cerebral ischemia in vivo and in vitro. We further identified let-7f as an upstream regulator of NDRG3 by the lentiviral transfection of rat cortical neurons and the dual luciferase analysis of human genes. In addition, a dual-color fluorescence in situ hybridization assay showed that when the expression of let-7f was elevated, the expression of NDRG3 mRNA was accordingly reduced in rat cerebral ischemia. Taken together, our results identify a new regulatory mechanism of let-7f on NDRG3 expression in the hypoxic response of cerebral ischemia and raise the possibility that the let-7f/NDRG3 pathway may serve as a potential target for the treatment of ischemic stroke.

Let-7a inhibits migration of melanoma cells via down-regulation of HMGA2 expression.

This study aimed to investigate the effects of exosomes derived from BM-MSCs transduced with let-7a on B16f10 cells and BM-MSCs. BM-MSCs were transduced with let-7a and the exosomes of them were isolated for further culture of B16f10 cells and BM-MSCs. The migration of B16f10 cells were detected by transwell, proliferation of B16f10 cells and BM-MSCs was examined by MTT method, HMGA2 expression was measured by western blot. In addition, the let-7a secreted level in exosomes and IGF level were measured by RT-PCR and ELISA respectively. Our results showed that the level of let-7a in exosomes derived from Let-7a-transducted BM-MSCs was increased after treated by exosomes. HMGA2 in B16f10 cells was down-regulated and cell survival rate of BM-MSCs was decreased. However, neither cell survival rate of B16f10 cells nor IGF-1 secreted by B16f10 cells in different groups had significant differences. In conclusion, Let-7a contained in exosomes can inhibit the migration of Melanoma cells and inhibit the proliferation of BM-MSCs.