RESUMO
Hereditary spastic paraplegias refer to a heterogeneous group of neurodegenerative disorders resulting from degeneration of the corticospinal tract. Clinical characterization of patients with hereditary spastic paraplegias represents progressive spasticity, exaggerated reflexes and muscular weakness. Here, to expand on the increasingly broad pools of previously unknown hereditary spastic paraplegia causative genes and subtypes, we performed whole exome sequencing for six affected and two unaffected individuals from two unrelated Chinese families with an autosomal dominant hereditary spastic paraplegia and lacking mutations in known hereditary spastic paraplegia implicated genes. The exome sequencing revealed two stop-gain mutations, c.247_248insGTGAATTC (p.I83Sfs*11) and c.526G>T (p.E176*), in the ubiquitin-associated protein 1 (UBAP1) gene, which co-segregated with the spastic paraplegia. We also identified two UBAP1 frameshift mutations, c.324_325delCA (p.H108Qfs*10) and c.425_426delAG (p.K143Sfs*15), in two unrelated families from an additional 38 Chinese pedigrees with autosomal dominant hereditary spastic paraplegias and lacking mutations in known causative genes. The primary disease presentation was a pure lower limb predominant spastic paraplegia. In vivo downregulation of Ubap1 in zebrafish causes abnormal organismal morphology, inhibited motor neuron outgrowth, decreased mobility, and shorter lifespan. UBAP1 is incorporated into endosomal sorting complexes required for transport complex I and binds ubiquitin to function in endosome sorting. Patient-derived truncated form(s) of UBAP1 cause aberrant endosome clustering, pronounced endosome enlargement, and cytoplasmic accumulation of ubiquitinated proteins in HeLa cells and wild-type mouse cortical neuron cultures. Biochemical and immunocytochemical experiments in cultured cortical neurons derived from transgenic Ubap1flox mice confirmed that disruption of UBAP1 leads to dysregulation of both early endosome processing and ubiquitinated protein sorting. Strikingly, deletion of Ubap1 promotes neurodegeneration, potentially mediated by apoptosis. Our study provides genetic and biochemical evidence that mutations in UBAP1 can cause pure autosomal dominant spastic paraplegia.
Assuntos
Proteínas de Transporte/genética , Paraplegia Espástica Hereditária/genética , Adolescente , Adulto , Animais , Povo Asiático/genética , Criança , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Linhagem , Peixe-ZebraRESUMO
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.
Assuntos
Encefalopatias/genética , Calcinose/genética , Predisposição Genética para Doença , Mutação , Doenças Neurodegenerativas/genética , Adulto , Idoso , Alelos , Transporte Biológico , Biomarcadores , Encefalopatias/diagnóstico , Encefalopatias/metabolismo , Calcinose/diagnóstico , Calcinose/metabolismo , Linhagem Celular Tumoral , China , Feminino , Genes sis , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/metabolismo , Neuroimagem , Fenótipo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptores Acoplados a Proteínas G/genética , Receptores Virais/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Tomografia Computadorizada por Raios X , Receptor do Retrovírus Politrópico e XenotrópicoRESUMO
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.
Assuntos
Doenças dos Gânglios da Base/diagnóstico , Doenças dos Gânglios da Base/genética , Calcinose/diagnóstico , Calcinose/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/genética , Deleção de Sequência , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Adolescente , Adulto , Idoso , Alelos , Criança , Feminino , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Linhagem , Fenótipo , Análise de Sequência de DNA , Receptor do Retrovírus Politrópico e Xenotrópico , Adulto JovemRESUMO
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.
Assuntos
Encefalopatias/genética , Encéfalo/metabolismo , Calcinose/genética , Receptores Acoplados a Proteínas G/genética , Receptores Virais/genética , Transcriptoma , Animais , Encéfalo/patologia , Encefalopatias/metabolismo , Encefalopatias/patologia , Calcinose/metabolismo , Calcinose/patologia , Expressão Gênica , Predisposição Genética para Doença , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mapas de Interação de Proteínas , RNA Mensageiro/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/análise , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores Acoplados a Proteínas G/análise , Receptores Acoplados a Proteínas G/metabolismo , Receptores Virais/análise , Receptores Virais/metabolismo , Regulação para Cima , Receptor do Retrovírus Politrópico e XenotrópicoRESUMO
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.
Assuntos
Povo Asiático/genética , Encéfalo/patologia , Calcinose/genética , Mutação/genética , Proteínas Proto-Oncogênicas c-sis/genética , Sequência de Aminoácidos , Sequência de Bases , Encéfalo/diagnóstico por imagem , Calcinose/diagnóstico por imagem , Família , Feminino , Humanos , Masculino , Linhagem , Proteínas Proto-Oncogênicas c-sis/química , Receptor do Retrovírus Politrópico e XenotrópicoRESUMO
OBJECTIVE: Expand genetic screening for atypical Type I sialidosis (ST-1) could address its underdiagnosed in both progressive myoclonic ataxia (PMA) and ataxia patients. To evaluate the potential founder effect of mutation in the population. METHODS: We enrolled 231 patients with PMA or ataxia from the First Affiliated Hospital of Fujian Medical University. Through Whole Exome Sequencing and Sanger sequencing, we identified the causative gene in patients. Haplotype analysis was employed to explore a potential founder effect of the NEU1 c.544A>G mutation. RESULTS: A total of 31 patients from 23 unrelated families were genetically diagnosed with ST-1. A significant 80.6% of these patients were homozygous for the c.544A>G mutation. We discovered six different NEU1 variants, including two novel mutations: c.951_968del and c.517T>G. The mean age of onset was 18.0 ± 7.1 years. The clinical spectrum of ST-1 featured ataxia and myoclonus as the most common initial symptoms. Over 40% suffered from controlled generalized tonic-clonic seizures. Mobility and independence varied greatly across the cohort. Cherry-red spots were rare, occurring in just 9.5% (2/21) of patients. Brain MRIs were typically unremarkable, except for two patients with unusual findings. EEGs showed diffuse paroxysmal activity in 17 patients. The c.544A>G mutation in NEU1 is a founder variant in Fujian, with a unique haplotype prevalent in East Asians. INTERPRETATION: ST-1 should be suspected in patients with PMA or ataxia in Southeast China, even without macular cherry-red spots and seizures, and the premier test could be a variant screening of the founder variant NEU1 c.544A>G.
RESUMO
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.
Assuntos
Calcinose , Modelos Animais de Doenças , Oligonucleotídeos Antissenso , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III , Animais , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo , Humanos , Camundongos , Calcinose/genética , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/administração & dosagem , Masculino , Feminino , Encefalopatias/genética , Encéfalo/metabolismo , Camundongos Transgênicos , Splicing de RNA/genética , Doenças dos Gânglios da Base , Doenças NeurodegenerativasRESUMO
In this study, three parameter optimization methods and two designs of experiments (DOE) were used for the optimization of three major design parameters ((bill diameter (D), billet length (L), and barrier wall design (BWD)) in crown forging to improve the formability of aluminum workpiece for shock absorbers. The first optimization method is the response surface method (RSM) combined with Box-Behnken's experimental design to establish fifteen (15) sets of parameter combinations for research. The second one is the main effects plot method (MEP). The third one is the multiobjective optimization method combined with Taguchi's experimental design method, which designed nine (9) parameter combinations and conducted research and analysis through grey relational analysis (GRA). Initially, a new type of forging die and billet in the controlled deformation zone (CDZ) was established by CAD (computer-aided design) modeling and the finite element method (FEM) for model simulation. Then, this investigation showed that the optimal parameter conditions obtained by these three optimization approaches (RSM, MEP, and multiobjective optimization) are consistent, with the same results. The best optimization parameters are the dimension of the billet ((D: 40 mm, the length of the billet (L): 205 mm, and the design of the barrier wall (BWD): 22 mm)). The results indicate that the optimization methods used in this research all have a high degree of accuracy. According to the research results of grey relational analysis (GRA), the size of the barrier wall design (BWD) in the controllable deformation zone (CDZ) has the greatest influence on the improvement of the preforming die, indicating that it is an important factor to increase the filling rate of aluminum crown forgings. At the end, the optimized parameters are verified by FEM simulation analysis and actual production validation as well as grain streamline distribution, processing map, and microstructure analysis on crown forgings. The novelty of this work is that it provides a novel preforming die through the mutual verification of different optimization methods to solve a typical problem such as material underfill.
RESUMO
Primary familial brain calcification (PFBC) is a neurogenetic disorder characterized by bilateral calcified deposits in the brain. We previously identified that MYORG as the first pathogenic gene for autosomal recessive PFBC, and established a Myorg-KO mouse model. However, Myorg-KO mice developed brain calcifications until nine months of age, which limits their utility as a facile PFBC model system. Hence, whether there is another typical animal model for mimicking PFBC phenotypes in an early stage still remained unknown. In this study, we profiled the mRNA expression pattern of myorg in zebrafish, and used a morpholino-mediated blocking strategy to knockdown myorg mRNA at splicing and translation initiation levels. We observed multiple calcifications throughout the brain by calcein staining at 2-4 days post-fertilization in myorg-deficient zebrafish, and rescued the calcification phenotype by replenishing myorg cDNA. Overall, we built a novel model for PFBC via knockdown of myorg by antisense oligonucleotides in zebrafish, which could shorten the observation period and replenish the Myorg-KO mouse model phenotype in mechanistic and therapeutic studies.
Assuntos
Encefalopatias , Calcinose , Doenças Neurodegenerativas , Animais , Encéfalo/metabolismo , Encefalopatias/genética , Calcinose/genética , Calcinose/metabolismo , Calcinose/patologia , Camundongos , Mutação , Doenças Neurodegenerativas/metabolismo , Linhagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra/genéticaRESUMO
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.
RESUMO
A novel bacterial strain HS0904 was isolated from a soil sample using 3,5-bis(trifluoromethyl) acetophenone as the sole carbon source. This bacterial isolate can asymmetrically reduce 3,5-bis(trifluoromethyl) acetophenone to (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol with high enantiometric excess (ee) value. Based on its morphological, physiological characteristics, Biolog, 16S rDNA sequence and phylogenetic analysis, strain HS0904 was identified as Leifsonia xyli HS0904. To our knowledge, this is the first reported case on the species L. xyli exhibited R-stereospecific carbonyl reductase and used for the preparation of chiral (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol. The optimization of parameters for microbial transformation of 3,5-bis(trifluoromethyl) acetophenone to (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol catalyzed by whole cells of L. xyli HS0904 was carried out by examining some key factors including buffer pH, reaction temperature, shaking speed, substrate concentration, and reaction time. The obtained optimized conditions for the bioreduction are as follows: buffer pH 8.0, 70 mM of 3,5-bis(trifluoromethyl) acetophenone, 100 g l(-1) of glucose as co-substrate, 200 g l(-1) of resting cells as biocatalyst, reaction for 30 h at 30 °C and 200 rpm. Under above conditions, 99.4% of product ee and best yield of 62% were obtained, respectively. The results indicated that isolate L. xyli HS0904 is a novel potential biocatalyst for the production of (1R)-[3,5-bis(trifluoromethyl)phenyl] ethanol.
Assuntos
Acetofenonas/metabolismo , Actinomycetales/classificação , Actinomycetales/metabolismo , Álcoois Benzílicos/metabolismo , Microbiologia do Solo , Actinomycetales/genética , Actinomycetales/isolamento & purificação , Técnicas de Tipagem Bacteriana , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Dados de Sequência Molecular , Oxirredução , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
BACKGROUND: Idiopathic basal ganglia calcification (IBGC) is a neurodegenerative disease characterized by symmetrical calcification of basal ganglia and other brain region, also known as Fahr's disease. It can be sporadic or familial, and there is no definite etiology at present. With the development of neuroimaging, the number of reports of IBGC has increased in recent years. However, due to its hidden onset, diverse clinical manifestations, and low incidence, it is likely to be misdiagnosed or ignored by potential patients and their family. CASE SUMMARY: We report a case of a 61-year-old man who presented with symptoms of dysphagia and alalia. His computed tomography scan of the brain revealed bilateral symmetric calcifications of basal ganglia, cerebellum, thalamus, and periventricular area. The genetic test showed a new mutation sites of MYORG, c.1438T>G mutation and c.1271_1272 TGGTGCGC insertion mutation. He was finally diagnosed with IBGC. CONCLUSION: It is important to detect MYORG mutation when IBGC is suspected, especially in those without an obvious family history, for better understanding of the underlying mechanism and identifying potential treatments.
RESUMO
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.
RESUMO
The original version of this article unfortunately contained mistakes in the affiliation section.
RESUMO
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.
Assuntos
Proteínas de Transporte/genética , Proteínas Mitocondriais/genética , Atrofia Óptica Hereditária de Leber/genética , Proteínas de Transporte/metabolismo , Criança , Feminino , Heterozigoto , Humanos , Proteínas Mitocondriais/metabolismo , Mutação , Atrofia Óptica Hereditária de Leber/patologia , Sequenciamento do ExomaRESUMO
BACKGROUND: Hereditary spastic paraplegias (HSP) is a heterogeneous group of rare neurodegenerative disorders affecting the corticospinal tracts. To date, more than 78 HSP loci have been mapped to cause HSP. However, both the clinical and mutational spectrum of Chinese patients with HSP remained unclear. In this study, we aim to perform a comprehensive analysis of clinical phenotypes and genetic distributions in a large cohort of Chinese HSP patients, and to elucidate the primary pathogenesis in this population. METHODS: We firstly performed next-generation sequencing targeting 149 genes correlated with HSP in 99 index cases of our cohort. Multiplex ligation-dependent probe amplification testing was further carried out among those patients without known disease-causing gene mutations. We simultaneously performed a retrospective study on the reported patients exhibiting HSP in other Chinese cohorts. All clinical and molecular characterization from above two groups of Chinese HSP patients were analyzed and summarized. Eventually, we further validated the cellular changes in fibroblasts of two major spastic paraplegia (SPG) patients (SPG4 and SPG11) in vitro. RESULTS: Most patients of ADHSP (94%) are pure forms, whereas most patients of ARHSP (78%) tend to be complicated forms. In ADHSP, we found that SPG4 (79%) was the most prevalent, followed by SPG3A (11%), SPG6 (4%) and SPG33 (2%). Subtle mutations were the common genetic cause for SPG4 patients and most of them located in AAA cassette domain of spastin protein. In ARHSP, the most common subtype was SPG11 (53%), followed by SPG5 (32%), SPG35 (6%) and SPG46 (3%). Moreover, haplotype analysis showed a unique haplotype was shared in 14 families carrying c.334C > T (p.R112*) mutation in CYP7B1 gene, suggesting the founder effect. Functionally, we observed significantly different patterns of mitochondrial dynamics and network, decreased mitochondrial membrane potential (Δψm), increased reactive oxygen species and reduced ATP content in SPG4 fibroblasts. Moreover, we also found the enlargement of LAMP1-positive organelles and abnormal accumulation of autolysosomes in SPG11 fibroblasts. CONCLUSIONS: Our study present a comprehensive clinical spectrum and genetic landscape for HSP in China. We have also provided additional evidences for mitochondrial and autolysosomal-mediated pathways in the pathogenesis of HSP.
Assuntos
Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/patologia , Adolescente , Adulto , Povo Asiático/genética , Criança , Pré-Escolar , China , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Fenótipo , Estudos Retrospectivos , Adulto JovemRESUMO
BACKGROUND: Progressive myoclonus epilepsies (PMEs) comprise a group of rare genetic disorders characterized by action myoclonus, epileptic seizures, and ataxia with progressive neurologic decline. Due to clinical and genetic heterogeneity of PMEs, it is difficult to decide which genes are affected. The aim of this study was to report an action myoclonus with or without renal failure syndrome (EPM4) family and summarize the clinical and genetic characteristics of all reported EPM4 patients. METHODS: In the present study, targeted next-generation sequencing (NGS) was applied to screen causative genes in a Chinese PME family. The candidate variant was further confirmed by cosegregation analysis and further functional analysis, including the reverse transcription polymerase chain reaction and Western blot of the proband's muscle. Moreover, literature data on the clinical and mutational features of all reported EPM4 patients were reviewed. RESULTS: The gene analysis revealed a novel homozygous splicing mutation (c.995-1G>A) of the SCARB2 gene in two brothers. Further functional analysis revealed that this mutation led to loss function of the SCARB2 protein. The classification of the candidate variant, according to the American College of Medical Genetics and Genomics standards and guidelines and functional analysis, was pathogenic. Therefore, these two brothers were finally diagnostically confirmed as EPM4. CONCLUSIONS: These present results suggest the potential for targeted NGS to conduct a more rapid and precise diagnosis for PME patients. A literature review revealed that mutations in the different functional domains of SCARB2 appear to be associated with the phenotype of EPM4.
Assuntos
Proteínas de Membrana Lisossomal/genética , Mutação , Epilepsias Mioclônicas Progressivas/genética , Receptores Depuradores/genética , Insuficiência Renal/genética , Humanos , Masculino , Epilepsias Mioclônicas Progressivas/complicações , Insuficiência Renal/complicaçõesRESUMO
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.
Assuntos
Astrócitos/metabolismo , Encefalopatias/genética , Calcinose/genética , Glicosídeo Hidrolases/genética , Mutação com Perda de Função , RNA Mensageiro/metabolismo , Adulto , Idoso , Alelos , Animais , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Mutação , LinhagemRESUMO
BACKGROUND: Until recently, primary familial brain calcification (PFBC) has been determined by four genes, SLC20A2, PDGFRB, PDGFB and XPR1. No studies have been carried out to analyze the gene mutation of PDGFB in Chinese population. OBJECTIVE: To screen mutations of PDGFB gene in a large cohort of Chinese PFBC patients with no SLC20A2 mutations. METHODS: We recruited 192 PFBC patients, including 21 index cases and 171 sporadic cases, in our study. Peripheral venous blood samples of all included participants were collected for genomic DNA extraction. The coding sequence of PDGFB was amplified by polymerase chain reaction (PCR) followed by direct sequencing. The potential effects of the identified variants on protein function were assessed by bioinformatics analysis. RESULTS: Three missense variants (c.35G>T, c.232C>T, and c.610C>A) and one nonsense variant (c.220G>T) of PDGFB were identified in five sporadic PFBC patients. The variant c.35G>T was found in 2 healthy controls from the same ethnic background, whereas c.220G>T, c.232C>T and c.610C>A were absent from 500 controls. c.220G>T (p.E74*) produced a stop codon in the place of the glutamicacid residue number 74. c.232C>T (p.R78C) occurred at highly conserved regions and were predicted as damaging by at least two computational predictive programs, suggesting that this variant were likely to have a causal role in PFBC. Although variant c.610C>A (p.P204T) also occurred at a highly conserved region, it was predicted to be most likely benign by two computational predictive programs, suggesting an uncertain role of this variant on PFBC. CONCLUSIONS: The present study identified one likely pathogenic variant (p.E74*) and two variants of uncertain significance (p.R78C and p.P204T) in PDGFB. Further studies of PDGF-B functional expression for these variants are still required to confirm the pathogenic effect.
RESUMO
To estimate the effect of ligustrazine on the expression of PECAM-1/CD31 and hematopoietic reconstitution in syngenic bone marrow transplanted mice, 56 BALB/c mice were divided into 3 groups: normal control group, BMT control group, ligustrazine treated group (BMT + Ligustrazine) and syngenic BMT mouse models were established according to the literatures. The BMT control group and the ligustrazine treated group were given orally 0.2 ml saline and 2 mg ligustrazine twice a day respectively. On days 7, 14, 21 after BMT, mice were killed and peripheral blood cells, bone marrow nucleated cells (BMNC) were detected. Histological observation of bone marrow was made and the CD31 expression was assayed by flow cytometry. The results showed that in ligustrazine treated group the peripheral blood cell, BMNC counts on days 7, 14, 21 after BMT were higher than in the BMT control group (P < 0.01 or P < 0.05). The expression of CD31 in ligustrazine treated group on days 7, 14, 21 after BMT was also higher than in the BMT control group (P < 0.01 or P < 0.05). In conclusion, ligustrazine enhances CD31 expression in bone marrow cells after syngenic bone marrow transplantation of mice, which may be related to the mechanisms underlying the ligustrazine accelerating hematopoietic reconstitution in syngeneic bone marrow transplantation.