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Primary familial brain calcification (PFBC) is a genetic neurological disease, yet no effective treatment is currently available. Here, we identified five novel intronic variants in SLC20A2 gene from six PFBC families. Three of these variants increased aberrant SLC20A2 pre-mRNA splicing by altering the binding affinity of splicing machineries to newly characterized cryptic exons, ultimately causing premature termination of SLC20A2 translation. Inhibiting the cryptic-exon incorporation with splice-switching ASOs increased the expression levels of functional SLC20A2 in cells carrying SLC20A2 mutations. Moreover, by knocking in a humanized SLC20A2 intron 2 sequence carrying a PFBC-associated intronic variant, the SLC20A2-KI mice exhibited increased inorganic phosphate (Pi) levels in cerebrospinal fluid (CSF) and progressive brain calcification. Intracerebroventricular administration of ASOs to these SLC20A2-KI mice reduced CSF Pi levels and suppressed brain calcification. Together, our findings expand the genetic etiology of PFBC and demonstrate ASO-mediated splice modulation as a potential therapy for PFBC patients with SLC20A2 haploinsufficiency.
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AIM: The effects of e-cigarettes on endothelial function remained controversial. The study aimed to investigate the effects of e-cigarettes on vascular endothelial function. METHODS AND RESULTS: PubMed, Web of Science, Embase, and Cochrane Library were searched up to December 2021. We only included the studies in which the control group included vaping without nicotine and tobacco. Pairwise and network meta-analyses were conducted for flow-mediated dilation (FMD), pulse wave velocity (PWV), and heart rate corrected augmentation index (AIx75). Eight studies involving 372 participants were eligible for this review. Compared with vaping without nicotine, e-cigarettes significantly increase in PWV (mean difference = 3.09; 95% confidential interval: 1.51-4.68, P < 0.001) and AIx75 (mean difference = 2.11; 95% confidential interval: 1.02-3.21, P < 0.001) indicators, but not affect FMD (mean difference = 0.78; 95% confidential interval: -0.08 to 1.64, P = 0.075). But compared with traditional tobacco, e-cigarettes did not affect FMD (mean difference = 0.28, 95% confidential interval: -0.45 to 0.59, P = 0.084). According to surface under the cumulative ranking curve (SUCRA), the e-cigarette ranked first for FMD (SUCRA = 97%), tobacco ranked first for PWV (SUCRA = 75%), and AIx75 (SUCRA = 99%). CONCLUSION: In summary, evidence from our pooled analyses indicated that acute inhalation of e-cigarettes leads to negative changes in vascular endothelial function. E-cigarettes cannot be used as an alternative to public health strategies for tobacco control and should not be considered cardiovascular safety products. More future research should be conducted to verify our findings.
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Sistemas Electrónicos de Liberación de Nicotina , Vapeo , Humanos , Nicotina/efectos adversos , Análisis de la Onda del Pulso , Ensayos Clínicos Controlados Aleatorios como Asunto , Vapeo/efectos adversosRESUMEN
Brain calcification is a critical aging-associated pathology and can cause multifaceted neurological symptoms. Cerebral phosphate homeostasis dysregulation, blood-brain barrier defects, and immune dysregulation have been implicated as major pathological processes in familial brain calcification (FBC). Here, we analyzed two brain calcification families and identified calcification co-segregated biallelic variants in the CMPK2 gene that disrupt mitochondrial functions. Transcriptome analysis of peripheral blood mononuclear cells (PBMCs) isolated from these patients showed impaired mitochondria-associated metabolism pathways. In situ hybridization and single-cell RNA sequencing revealed robust Cmpk2 expression in neurons and vascular endothelial cells (vECs), two cell types with high energy expenditure in the brain. The neurons in Cmpk2-knockout (KO) mice have fewer mitochondrial DNA copies, down-regulated mitochondrial proteins, reduced ATP production, and elevated intracellular inorganic phosphate (Pi) level, recapitulating the mitochondrial dysfunction observed in the PBMCs isolated from the FBC patients. Morphologically, the cristae architecture of the Cmpk2-KO murine neurons was also impaired. Notably, calcification developed in a progressive manner in the homozygous Cmpk2-KO mice thalamus region as well as in the Cmpk2-knock-in mice bearing the patient mutation, thus phenocopying the calcification pathology observed in the patients. Together, our study identifies biallelic variants of CMPK2 as novel genetic factors for FBC; and demonstrates how CMPK2 deficiency alters mitochondrial structures and functions, thereby highlighting the mitochondria dysregulation as a critical pathogenic mechanism underlying brain calcification.
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BACKGROUND: Grazing disturbance usually affects floral display and pollination efficiency in the desert steppe, which may cause pollen limitation in insect-pollinated plants. Effective pollination is essential for the reproductive success of insect-pollinated plants and insufficient pollen transfer may result in pollen limitation. Caragana microphylla Lam is an arid region shrub with ecological importance. Few studies have been conducted on how grazing disturbance influences pollen limitation and pollination efficiency of C. microphylla. Here, we quantify the effect of different grazing intensities on floral display, pollinator visitation frequency and seed production in the Urat desert steppe. RESULTS: In C. microphylla, supplemental hand pollination increased the seed set, and pollen limitation was the predominant limiting factor. As the heavy grazing significantly reduced the seed set in plants that underwent open-pollination, but there was no significant difference in the seed set between plants in the control plots and plants in the moderate grazing plots. Furthermore, there was a higher pollinator visitation frequency in plants in the control plots than in plants in the heavy grazing plots. CONCLUSIONS: We found that pollinator visitation frequency was significantly associated with the number of open flowers. Our findings also demonstrated that seed production is associated with pollinator visitation frequency, as indicated by increased seed production in flowers with higher pollinator visitation frequency. Therefore, this study provides insight into the effect of different grazing intensities on floral display that are important for influencing pollinator visitation frequency and pollination efficiency in desert steppes.
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Flores , Herbivoria , Insectos , Polen , Polinización , Animales , Flores/fisiología , Insectos/fisiología , Plantas/parasitología , Polinización/fisiología , Clima Desértico , Herbivoria/fisiologíaAsunto(s)
Enfermedades Autoinmunes del Sistema Nervioso , Calcinosis , Malformaciones del Sistema Nervioso , Enfermedades Autoinmunes del Sistema Nervioso/complicaciones , Enfermedades Autoinmunes del Sistema Nervioso/diagnóstico por imagen , Enfermedades Autoinmunes del Sistema Nervioso/genética , Calcinosis/complicaciones , Calcinosis/diagnóstico por imagen , Calcinosis/genética , Humanos , Malformaciones del Sistema Nervioso/complicaciones , Malformaciones del Sistema Nervioso/diagnóstico por imagen , Malformaciones del Sistema Nervioso/genéticaRESUMEN
Primary familial brain calcification (PFBC) is a progressive neurological disorder manifesting as bilateral brain calcifications in CT scan with symptoms as parkinsonism, dystonia, ataxia, psychiatric symptoms, etc. Recently, pathogenic variants in MYORG have been linked to autosomal recessive PFBC. This study aims to elucidate the mutational and clinical spectrum of MYORG mutations in a large cohort of Chinese PFBC patients with possible autosomal recessive or absent family history. Mutational analyses of MYORG were performed by Sanger sequencing in a cohort of 245 PFBC patients including 21 subjects from 10 families compatible with a possibly autosomal-recessive trait and 224 apparently sporadic cases. In-depth phenotyping and neuroimaging features were investigated in all patients with novel MYORG variants. Two nonsense variants (c.442C > T, p. Q148*; c.972C > A, p. Y324*) and two missense variants (c.1969G>C, p. G657R; c.2033C > G, p. P678R) of MYORG were identified in four sporadic PFBC patients, respectively. These four novel variants were absent in gnomAD, and their amino acid were highly conserved, suggesting these variants have a pathogenic impact. Patients with MYORG variants tend to display a homogeneous clinical spectrum, showing extensive brain calcification and parkinsonism, dysarthria, ataxia, or vertigo. Our findings supported the pathogenic role of MYORG variants in PFBC and identified two pathogenic variants (c.442C > T, c.972C > A), one likely pathogenic variant (c.2033C > G), and one variant of uncertain significance (c.1969G>C), further expanding the genetic and phenotypic spectrum of PFBC-MYORG.
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YgfY(SdhE/CptB) is highly conserved while has controversial functions in bacteria. It works as an antitoxin and composes a type IV toxin-antitoxin system with YgfX(CptA) typically in Escherichia coli, while functions as an flavinylation factor of succinate dehydrogenase and fumarate reductase typically in Serratia sp. In this study, we report the contribution of YgfY in Shewanella oneidensis MR-1 to tolerance of low temperature and nitrite. YgfY deficiency causes several growth defects of S. oneidensis MR-1 at low temperature, while YgfX do not cause a growth defect or morphological change of S. oneidensis MR1-1 and E. coli. YgfY do not interact with FtsZ and MreB nor with YgfX examined by bacterial two-hybrid assay. YgfY effect on growth under low temperature is not attributed to succinate dehydrogenase (SDH) because a mutant without SDH grows comparably with the wild-type strain in the presence of succinate. The ygfY mutant shows impaired tolerance to nitrite. Transcription of nitrite reductase and most ribosome proteins is significantly decreased in the ygfY mutant, which is consistent with the phenotypes detected above. Effects of YgfY on growth and nitrite tolerance are closely related to the RGXXE motif in YgfY. In summary, this study demonstrates pleiotropic impacts of YgfY in S. oneidensis MR-1, and sheds a light on the physiological versatility of YgfY in bacteria.
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Glycyrrhetinic acid (GA), a hydrolysate of glycyrrhizic acid from licorice root extract, has been used to treat liver fibrotic diseases. However, the molecular mechanism involved in the antifibrotic effects of GA remains unclear. The involvement of miR-663a and its roles in TGF-ß-1-induced hepatic stellate cell (HSC) activation remains unclear. In this study, we investigated the roles of miR-663a in the activation of HSCs and the antifibrosis mechanism of GA. MiR-663a expression was downregulated in TGF-ß-treated HSCs. The overexpression of miR-663a inhibited HSC proliferation. TGF-ß-1was confirmed as a direct target gene of miR-663a. MiR-663a alleviated HSC activation, concomitant with decreased expression of α-smooth muscle actin (α-SMA), human α2 (I) collagen (COL1A2), TGF-ß1, TGF-ßRI, Smad4, p-Smad2, and p-Smad3. GA upregulated miR-663a expression and inhibited the TGF-ß/Smad pathway in HSCs. Further studies showed that miR-663a inhibitor treatment reversed GA-mediated downregulation of TGF-ß1, TGF-ßRI, Smad4, p-Smad2, p-Smad3, α-SMA, and CoL1A2 in TGF-ß1-treated HSCs. These results show that miR-663a suppresses HSC proliferation and activation and the TGF-ß/Smad signaling pathway, highlighting that miR-663a can be utilized as a therapeutic target for hepatic fibrosis. GA inhibits, at least in part, HSC proliferation and activation via targeting the miR-663a/TGF-ß/Smad signaling pathway.
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The original version of this article unfortunately contained mistakes in the affiliation section.
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We here report a genome-editing strategy to correct spinal muscular atrophy (SMA). Rather than directly targeting the pathogenic exonic mutations, our strategy employed Cas9 and guide-sgRNA for the targeted disruption of intronic splicing-regulatory elements. We disrupted intronic splicing silencers (ISSs, including ISS-N1 and ISS + 100) of survival motor neuron (SMN) 2, a key modifier gene of SMA, to enhance exon 7 inclusion and full-length SMN expression in SMA iPSCs. Survival of splicing-corrected iPSC-derived motor neurons was rescued with SMN restoration. Furthermore, co-injection of Cas9 mRNA from Streptococcus pyogenes (SpCas9) or Cas9 from Staphylococcus aureus (SaCas9) alongside their corresponding sgRNAs targeting ISS-N1 into zygotes rescued 56% and 100% of severe SMA transgenic mice (Smn -/-, SMN2 tg/-). The median survival of the resulting mice was extended to >400 days. Collectively, our study provides proof-of-principle for a new strategy to therapeutically intervene in SMA and other RNA-splicing-related diseases.
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Autosomal recessive optic neuropathies (IONs) are extremely rare disorders affecting retinal ganglion cells and the nervous system. RTN4IP1 has recently been identified as the third known gene associated with the autosomal recessive ION optic atrophy 10 (OPA10). Patients with RTN4IP1 mutations show early-onset optic neuropathy that can be followed by additional neurological symptoms such as seizures, ataxia, mental retardation, or even severe encephalopathy. Here, we report two siblings from a Chinese family who presented with early-onset optic neuropathy, epilepsy, and mild intellectual disability. Using whole exome sequencing combined with Sanger sequencing, we identified novel compound heterozygous RTN4IP1 mutations (c.646G > A, p.G216R and c.1162C > T, p.R388X) which both co-segregated with the disease phenotype and were predicted to be disease-causing by prediction software. An in vitro functional study in urine cells obtained from one of the patients revealed low expression of the RTN4IP1 protein. Our results identify novel compound heterozygous mutations in RTN4IP1 which are associated with OPA10, highlighting the frequency of RTN4IP1 mutations in human autosomal recessive IONs. To our knowledge, this is the first report of RTN4IP1 carriers from China.
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Proteínas Portadoras/genética , Proteínas Mitocondriales/genética , Atrofia Óptica Hereditaria de Leber/genética , Proteínas Portadoras/metabolismo , Niño , Femenino , Heterocigoto , Humanos , Proteínas Mitocondriales/metabolismo , Mutación , Atrofia Óptica Hereditaria de Leber/patología , Secuenciación del ExomaRESUMEN
Primary familial brain calcification (PFBC) is a rare neurological disorder. Mutations in five genes (SLC20A2, PDGFRB, PDGFB, XPR1, and MYORG) have been linked to PFBC. Here, we used SYBR green-based real-time quantitative polymerase chain reaction (PCR) assay and denaturing high-performance liquid chromatography analysis to detect copy number variants (CNVs) in 20 unrelated patients with PFBC, negatively sequenced for the five known genes. We identified three deletions in SLC20A2, including a large de novo full gene deletion and two exonic deletions confined to exon 2 and exon 6, respectively. Subsequent linked-read whole-genome sequencing of the patient with the large deletion showed a 1.7 Mb heterozygous deletion which removed the entire coding regions of SLC20A2 as well as 21 other genes. In the family with a deletion of exon 6, a missense variant of uncertain significance (SLC20A2: p.E267Q) also co-segregated with the disease. Functional assay showed the deletion could result in significantly impaired phosphate transport, whereas the p.E267Q variant did not. Our results confirm that deletion in SLC20A2 is a causal mechanism for PFBC and highlight the importance of functional study for classifying a rare missense variant as (likely) pathogenic.
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Enfermedades de los Ganglios Basales/diagnóstico , Enfermedades de los Ganglios Basales/genética , Calcinosis/diagnóstico , Calcinosis/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Enfermedades Neurodegenerativas/diagnóstico , Enfermedades Neurodegenerativas/genética , Eliminación de Secuencia , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/genética , Adolescente , Adulto , Anciano , Alelos , Niño , Femenino , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Repeticiones de Microsatélite , Persona de Mediana Edad , Linaje , Fenotipo , Análisis de Secuencia de ADN , Receptor de Retrovirus Xenotrópico y Politrópico , Adulto JovenRESUMEN
Primary familial brain calcification (PFBC) is a rare neurodegenerative disorder with four causative genes (SLC20A2, PDGFRB, PDGFB, and XPR1) that have been identified. Here, we aim to describe the mutational spectrum of four causative genes in a series of 226 unrelated Chinese PFBC patients. Mutations in four causative genes were detected in 16.8% (38/226) of PFBC patients. SLC20A2 mutations accounted for 14.2% (32/226) of all patients. Mutations in the other three genes were relatively rare, accounting for 0.9% (2/226) of all patients, respectively. Clinically, 44.8% of genetically confirmed patients (probands and relatives) were considered symptomatic. The most frequent symptoms were chronic headache, followed by movement disorders and vertigo. Moreover, the total calcification score was significantly higher in the symptomatic group compared to the asymptomatic group. Functionally, we observed impaired phosphate transport induced by seven novel missense mutations in SLC20A2 and two novel mutations in XPR1. The mutation p.D164Y in XPR1 might result in low protein expression through an enhanced proteasome pathway. In conclusion, our study further confirms that mutations in SLC20A2 are the major cause of PFBC and provides additional evidence for the crucial roles of phosphate transport impairment in the pathogenies of PFBC.
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Encefalopatías/genética , Calcinosis/genética , Predisposición Genética a la Enfermedad , Mutación , Enfermedades Neurodegenerativas/genética , Adulto , Anciano , Alelos , Transporte Biológico , Biomarcadores , Encefalopatías/diagnóstico , Encefalopatías/metabolismo , Calcinosis/diagnóstico , Calcinosis/metabolismo , Línea Celular Tumoral , China , Femenino , Genes sis , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Enfermedades Neurodegenerativas/diagnóstico , Enfermedades Neurodegenerativas/metabolismo , Neuroimagen , Fenotipo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptores Acoplados a Proteínas G/genética , Receptores Virales/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/genética , Tomografía Computarizada por Rayos X , Receptor de Retrovirus Xenotrópico y PolitrópicoRESUMEN
Primary familial brain calcification (PFBC) is a genetically heterogeneous disorder characterized by bilateral calcifications in the basal ganglia and other brain regions. The genetic basis of this disorder remains unknown in a significant portion of familial cases. Here, we reported a recessive causal gene, MYORG, for PFBC. Compound heterozygous or homozygous mutations of MYORG co-segregated completely with PFBC in six families, with logarithm of odds (LOD) score of 4.91 at the zero recombination fraction. In mice, Myorg mRNA was expressed specifically in S100ß-positive astrocytes, and knockout of Myorg induced the formation of brain calcification at 9 months of age. Our findings provide strong evidence that loss-of-function mutations of MYORG cause brain calcification in humans and mice.
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Astrocitos/metabolismo , Encefalopatías/genética , Calcinosis/genética , Glicósido Hidrolasas/genética , Mutación con Pérdida de Función , ARN Mensajero/metabolismo , Adulto , Anciano , Alelos , Animales , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Mutación , LinajeRESUMEN
Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder caused by survival motor neuron (SMN) protein deficiency leading the loss of motor neurons in the anterior horns of the spinal cord and brainstem. More than 95% of SMA patients are attributed to the homozygous deletion of survival motor neuron 1 (SMN1) gene, and approximately 5% are caused by compound heterozygous with a SMN1 deletion and a subtle mutation. Here, we identified a rare variant c.835-5T>G in intron 6 of SMN1 in a patient affected with type I SMA. We analyzed the functional consequences of this mutation on mRNA splicing in vitro. After transfecting pCI-SMN1, pCI-SMN2, and pCI-SMN1 c.835-5T>G minigenes into HEK293, Neuro-2a, and SHSY5Y cells, reverse transcription polymerase chain reaction (RT-PCR) was performed to compare the splicing effects of these minigenes. Finally, we found that this mutation resulted in the skipping of exon 7 in SMN1, which confirmed the genetic diagnosis of SMA.
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Atrofia Muscular Espinal/genética , Mutación , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Línea Celular Tumoral , Células HEK293 , Humanos , Lactante , Masculino , Atrofia Muscular Espinal/patología , Empalme del ARN , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismoRESUMEN
Circulating microRNAs (miRNAs) can be employed as biomarkers to diagnose liver and other diseases. Noninvasive approaches are needed to complement and improve the current strategies for screening for biomarkers liver cirrhosis. We determined whether the serum levels of miRNAs can distinguish between chronic hepatitis B (CHB) and CHB-induced cirrhosis (HBC) and investigated the potential mechanisms involved. We found that serum miR-27a was significantly up-regulated in HBC, distinguishing HBC from CHB and healthy controls (Ctrl) (P<0.0001, the area of under the curve (AUC) =0.82 and 0.87, respectively). Specifically, when miR-27a was combined with miR-122, HBC was differentiated from CHB with an AUC=0.94. The serum miR-27a level in HBC patients with hepatic decompensation was significantly higher than that in patients with compensated HBC (P=0.0009). MiR-27a was also significantly up-regulated in the serum of rats with DMN-induced liver cirrhosis compared to that in saline-treated rats (P<0.0001). Furthermore, the down-regulation of miR-27a inhibited the proliferation and overexpression of miR-27a in activated hepatic stellate cells (HSCs) through the up-regulation of α-SMA and COL1A2 expression by targeting PPARγ, FOXO1, APC, P53 and RXRα. Our study demonstrated that circulating miR-27a can be used as a predictor for the activation of HSCs and the occurrence and development of HBC.
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Spinal muscular atrophy (SMA) is a lethal childhood neurodegenerative disorder that is caused by the homozygous deletion of survival motor neuron 1 (SMN1). To date, no effective treatments are available. In the current study, urine cells taken from SMA patients were cultured and the application of patient-derived urine cells was determined in drug intervention. A total of 13 SMA patient-derived urine cell lines and 40 control cell lines were established. SMN was highly expressed in the nucleus and cytoplasm. Patient-derived urine cells expressed low levels of SMN protein compared with controls, they exhibited good tolerance to chemical and electrical damage. SMN expression was upregulated following treatment with histone deacetylase inhibitors and the effect was greater in groups treated with morpholino modified antisense oligo, which targets ISS-N1 in SMN2 intron 7. The results of the current study indicated that SMA patient-derived urine cells may be useful in the initial screening of potential compounds and drugs to treat SMA.
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Primary familial brain calcification (PFBC) is a neuropsychiatric disorder characterized by bilateral cerebral calcification with diverse neurologic or psychiatric symptoms. Recently, XPR1 variation has accounted for PFBC as another new causative gene. However, little is known about the distribution and basic function of XPR1 and its interaction with the other three pathogenic genes for PFBC (SLC20A2, PDGFRB and PDGFB). The aim of this study was to further clarify the role of XPR1 in PFBC brain pathology. As a result, gene expression profiles showed that XPR1 mRNA was widely expressed throughout the mouse brain. Cerebellum and striatum, most commonly affected in PFBC, contained a higher level of XPR1 protein than other brain regions. Additionally, XPR1 deficiency seriously affected Pi efflux and XPR1 mutations seemed to have an effect through haploinsufficiency mechanism. The immunoprecipitation and immunohistochemical studies demonstrated that XPR1 could interact with PDGFRB and might form a complex on the cell membrane. These results suggested that XPR1 played a fundamental role in the maintenance of cellular phosphate balance in the brain. This provided us with a novel perspective on understanding the pathophysiology of PFBC. The expression networks and interaction with the known pathogenic genes could shed new light on additional candidate genes for PFBC.
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Encefalopatías/genética , Encéfalo/metabolismo , Calcinosis/genética , Receptores Acoplados a Proteínas G/genética , Receptores Virales/genética , Transcriptoma , Animales , Encéfalo/patología , Encefalopatías/metabolismo , Encefalopatías/patología , Calcinosis/metabolismo , Calcinosis/patología , Expresión Génica , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Mapas de Interacción de Proteínas , ARN Mensajero/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/análisis , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores Acoplados a Proteínas G/análisis , Receptores Acoplados a Proteínas G/metabolismo , Receptores Virales/análisis , Receptores Virales/metabolismo , Regulación hacia Arriba , Receptor de Retrovirus Xenotrópico y PolitrópicoRESUMEN
Perfluorooctane sulfonate (PFOS), a new kind of persistent organic pollutant, is widely distributed in the environment and exists in various organisms, where it is also a neurotoxic compound. However, the potential mechanism of its neurotoxicity is still unclear. To examine the role of epigenetics in the neurotoxicity induced by PFOS, SK-N-SH cells were treated with different concentrations of PFOS or control medium (0.1% DMSO) for 48 h. The mRNA levels of DNA methyltransferases (DNMTs) and Brain-derived neurotrophic factor (BDNF), microRNA-16, microRNA-22, and microRNA-30a-5p were detected by Quantitative PCR (QPCR). Enzyme Linked Immunosorbent Assay (ELISA) was used to measure the protein levels of BDNF, and a western blot was applied to analyze the protein levels of DNMTs. Bisulfite sequencing PCR (BSP) was used to detect the methylation status of the BDNF promoter I and IV. Results of MTT assays indicated that treatment with PFOS could lead to a significant decrease of cell viability, and the treated cells became shrunk. In addition, PFOS exposure decreased the expression of BDNF at mRNA and protein levels, increased the expression of microRNA-16, microRNA-22, microRNA-30a-5p, and decreased the expression of DNMT1 at mRNA and protein levels, but increased the expression of DNMT3b at mRNA and protein levels. Our results also demonstrate that PFOS exposure changes the methylation status of BDNF promoter I and IV. The findings of the present study suggest that methylation regulation of BDNF gene promoter and increases of BDNF-related-microRNA might underlie the mechanisms of PFOS-induced neurotoxicity.
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Ácidos Alcanesulfónicos/toxicidad , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Contaminantes Ambientales/toxicidad , Epigénesis Genética/efectos de los fármacos , Fluorocarburos/toxicidad , Factor Neurotrófico Derivado del Encéfalo/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , ADN-Citosina Metilasas/genética , ADN-Citosina Metilasas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Expresión Génica/efectos de los fármacos , Humanos , MicroARNs/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Four causative genes, including solute carrier family 20 member 2 (SLC20A2), platelet-derived growth factor receptor b (PDGFRB), platelet-derived growth factor b (PDGFB)and xenotropic and polytropic retrovirus receptor 1 (XPR1), have been identified to cause primary familial brain calcification (PFBC). However, PDGFRB mutations seem to be quite rare and no PDGFRB mutations have been reported in Chinese PFBC patients. A total of 146 PFBC patients including 12 families and 134 sporadic patients were recruited in this study. All of them were previously tested negative for the SLC20A2. Mutational analyses of the entire exons and exon-intron boundaries of PDGFRB were carried out by direct gene sequencing. In silico analyses of the identified variants were conducted using Mutation Taster, PolyPhen-2 and Sorts Intolerant From Tolerant. Two heterozygous variants, c.3G>A and c.2209G>A, of the PDGFRB gene were revealed in two PFBC families, respectively. These two variants were not observed in 200 healthy controls. The variant c.3G>A was located in exon 2 and affected the initiation codon of the PDGFRB gene. The variant c.2209G>A resulted in amino-acid substitutions of aspartic acid to asparagine at position 737. Both of these two variants co-segregated with the disease phenotype (variant carriers in Family 1: I1, II2 and II3; variant carriers in Family 2: I2 and II8), suggesting a pathogenic impact of these variants. The prevalence of PDGFRB mutations in Chinese PFBC patients seems to be quite low, indicating that PDGFRB is not a major causative gene of PFBC in Chinese population.