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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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BACKGROUND: Intronic GAA repeat expansion ([GAA] ≥250) in FGF14 is associated with the late-onset neurodegenerative disorder, spinocerebellar ataxia 27B (SCA27B, GAA-FGF14 ataxia). We aim to determine the prevalence of the GAA repeat expansion in FGF14 in Chinese populations presenting late-onset cerebellar ataxia (LOCA) and evaluate the characteristics of tandem repeat inheritance, radiological features and sympathetic nerve involvement. METHODS: GAA-FGF14 repeat expansion was screened in an undiagnosed LOCA cohort (n = 664) and variations in repeat-length were analyzed in families of confirmed GAA-FGF14 ataxia patients. Brain magnetic resonance imaging (MRI) was used to evaluate the radiological feature in GAA-FGF14 ataxia patients. Clinical examinations and sympathetic skin response (SSR) recordings in GAA-FGF14 patients (n = 16) were used to quantify sympathetic nerve involvement. RESULTS: Two unrelated probands (2/664) were identified. Genetic screening for GAA-FGF14 repeat expansion was performed in 39 family members, 16 of whom were genetically diagnosed with GAA-FGF14 ataxia. Familial screening revealed expansion of GAA repeats in maternal transmissions, but contraction upon paternal transmission. Brain MRI showed slight to moderate cerebellar atrophy. SSR amplitude was lower in GAA-FGF14 patients in pre-symptomatic stage compared to healthy controls, and further decreased in the symptomatic stage. CONCLUSIONS: GAA-FGF14 ataxia was rare among Chinese LOCA cases. Parental gender appears to affect variability in GAA repeat number between generations. Reduced SSR amplitude is a prominent feature in GAA-FGF14 patients, even in the pre-symptomatic stage.
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BACKGROUND: Hereditary spastic paraplegias (HSP) are neurologic disorders characterized by progressive lower-extremity spasticity. Despite the identification of several HSP-related genes, many patients lack a genetic diagnosis. OBJECTIVES: The aims were to confirm the pathogenic role of biallelic COQ4 mutations in HSP and elucidate the clinical, genetic, and functional molecular features of COQ4-associated HSP. METHODS: Whole exome sequences of 310 index patients with HSP of unknown cause from three distinct populations were analyzed to identify potential HSP causal genes. Clinical data obtained from patients harboring candidate causal mutations were examined. Functional characterization of COQ4 variants was performed using bioinformatic tools, single-cell RNA sequencing, biochemical assays in cell lines, primary fibroblasts, induced pluripotent stem cell-derived pyramidal neurons, and zebrafish. RESULTS: Compound heterozygous variants in COQ4, which cosegregated with HSP in pedigrees, were identified in 7 patients from six unrelated families. Patients from four of the six families presented with pure HSP, whereas probands of the other two families exhibited complicated HSP with epilepsy or with cerebellar ataxia. In patient-derived fibroblasts and COQ4 knockout complementation lines, stable expression of these missense variants exerted loss-of-function effects, including mitochondrial reactive oxygen species accumulation, decreased mitochondrial membrane potential, and lower ubiquinone biosynthesis. Whereas differentiated pyramidal neurons expressed high COQ4 levels, coq4 knockdown zebrafish displayed severe motor dysfunction, reflecting motor neuron dysregulation. CONCLUSIONS: Our study confirms that loss-of-function, compound heterozygous, pathogenic COQ4 variants are causal for autosomal recessive pure and complicated HSP. Moreover, reduced COQ4 levels attributable to variants correspond with decreased ubiquinone biosynthesis, impaired mitochondrial function, and higher phenotypic disease severity. © 2023 International Parkinson and Movement Disorder Society.
Subject(s)
Spastic Paraplegia, Hereditary , Zebrafish , Animals , Humans , Ubiquinone/genetics , Spastic Paraplegia, Hereditary/genetics , Mutation/genetics , Mutation, Missense , Mitochondrial Proteins/geneticsABSTRACT
CGG repeat expansions in LOC642361/NUTM2B-AS1 have recently been identified as a cause of oculopharyngeal myopathy with leukoencephalopathy. However, since only three patients from a single family were reported, it remains unknown whether their clinicopathological features are typical for CGG repeat expansions in LOC642361/NUTM2B-AS1. Here, using repeat-primed-polymerase chain reaction and long-read sequencing, we identify 12 individuals from 3 unrelated families with CGG repeat expansions in LOC642361/NUTM2B-AS1, typically presenting with oculopharyngodistal myopathy. The CGG repeat expansions range from 161 to 669 repeat units. Most of the patients present with ptosis, restricted eye movements, dysphagia, dysarthria, and diffuse limb muscle weakness. Only one patient shows T2-weighted hyperintensity in the cerebellar white matter surrounding the deep cerebellar nuclei on brain magnetic resonance imaging. Muscle biopsies from three patients show a myopathic pattern and rimmed vacuoles. Analyses of muscle biopsies suggest that CGG repeat expansions in LOC642361/NUTM2B-AS1 may deleteriously affect aggrephagic capacity, suggesting that RNA toxicity and mitochondrial dysfunction may contribute to pathogenesis. Our study thus expands the phenotypic spectrum for the CGG repeat expansion of LOC642361/NUTM2B-AS1 and indicates that this genetic variant typically manifests as oculopharyngodistal myopathy with chronic myopathic changes with rimmed vacuoles and filamentous intranuclear inclusions in muscle fibers.
Subject(s)
Muscular Diseases , Muscular Dystrophies , Humans , Muscle Weakness , Muscular Diseases/genetics , Muscular Diseases/pathology , Muscular Dystrophies/genetics , Muscular Dystrophies/pathologyABSTRACT
BACKGROUND: Yanghe Watershed has low annual rainfall, uneven spatial and temporal distribution, extreme shortage of water resources in some areas. The contradiction between supply and demand of water for agricultural production is prominent and the expected production value cannot be achieved. Therefore, it is necessary to investigate the supply and demand of agricultural water resources and the impact of green water on agricultural crops in Yanghe Watershed. RESULTS: This article proposes a new crop economic model for increasing the green-water footprint to blue-water footprint ratio (GWF:BWF) in accordance with the regional characteristics, alleviating agricultural water shortage in irrigation areas, optimizing water resource allocation, and achieving sustainable agricultural development. The proposition is based on a study of five crops in eight districts and counties in the Yanghe River watershed. By combining the economic model F with a crop water production function, we achieved 89.3%, 88.9%, 97.1%, 81.5%, and 87.0% of the optimal water demands of the five crops, respectively, and effectively improved the underground irrigation of crops and the water resource utilization efficiency. CONCLUSION: The GWF:BWF threshold interval was subsequently selected based on the temporal changes in the BWF and GWF in the study area. This enabled significant reduction of the planting area of blue-water crops and increase in the proportion of green-water crops, while also improving the agricultural economy of the Yanghe Watershed. The proposed model promises to afford enhanced management of agricultural irrigation areas that experience rainfall shortage. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Subject(s)
Water Resources , Water , Water/analysis , Agriculture , Agricultural Irrigation , Crops, Agricultural , China , Water SupplyABSTRACT
OBJECTIVE: Despite the increasing number of genes associated with Charcot-Marie-Tooth (CMT) disease, many patients currently still lack appropriate genetic diagnosis for this disease. Autosomal dominant mutations in aminoacyl-tRNA synthetases (ARSs) have been implicated in CMT. Here, we describe causal missense mutations in the gene encoding seryl-tRNA synthetase 1 (SerRS) for 3 families affected with CMT. METHODS: Whole-exome sequencing was performed in 16 patients and 14 unaffected members of 3 unrelated families. The functional impact of the genetic variants identified was investigated using bioinformatic prediction tools and confirmed using cellular and biochemical assays. RESULTS: Combined linkage analysis for the 3 families revealed significant linkage (Zmax LOD = 6.9) between the genomic co-ordinates on chromosome 1: 108681600-110300504. Within the linkage region, heterozygous SerRS missense variants segregated with the clinical phenotype in the 3 families. The mutant SerRS proteins exhibited reduced aminoacylation activity and abnormal SerRS dimerization, which suggests the impairment of total protein synthesis and induction of eIF2α phosphorylation. INTERPRETATION: Our findings suggest the heterozygous SerRS variants identified represent a novel cause for autosomal dominant CMT. Mutant SerRS proteins are known to impact various molecular and cellular functions. Our findings provide significant advances on the current understanding of the molecular mechanisms associated with ARS-related CMT. ANN NEUROL 2023;93:244-256.
Subject(s)
Charcot-Marie-Tooth Disease , Serine-tRNA Ligase , Humans , Charcot-Marie-Tooth Disease/genetics , Charcot-Marie-Tooth Disease/metabolism , Serine-tRNA Ligase/genetics , Mutation , Heterozygote , Mutation, Missense/geneticsABSTRACT
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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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.
Subject(s)
Brain Diseases , Calcinosis , Neurodegenerative Diseases , Animals , Brain/metabolism , Brain Diseases/genetics , Calcinosis/genetics , Calcinosis/metabolism , Calcinosis/pathology , Mice , Mutation , Neurodegenerative Diseases/metabolism , Pedigree , RNA, Messenger/genetics , RNA, Messenger/metabolism , Zebrafish/geneticsABSTRACT
OBJECTIVE: Oculopharyngodistal myopathy (OPDM) is an adult-onset neuromuscular disease characterized by progressive ptosis, dysarthria, ophthalmoplegia, and distal muscle weakness. Recent studies revealed that GGC repeat expansions in 5'-UTR of LRP12, GIPC1, and NOTCH2NLC are associated with OPDM. Despite these advances, approximately 30% of OPDM patients remain genetically undiagnosed. Herein, we aim to investigate the genetic basis for undiagnosed OPDM patients in two unrelated Chinese Han families. METHODS: Parametric linkage analysis was performed. Long-read sequencing followed by repeat-primed polymerase chain reaction and amplicon length polymerase chain reaction were used to determine the genetic cause. Targeted methylation sequencing was implemented to detect epigenetic changes. The possible pathogenesis mechanism was investigated by quantitative polymerase chain reaction, immunoblotting, RNA fluorescence in situ hybridization, and immunofluorescence staining of muscle biopsy samples. RESULTS: The disease locus was mapped to 12q24.3. Subsequently, GGC repeat expansion in the promoter region of RILPL1 was identified in six OPDM patients from two families, findings consistent with a founder effect, designated as OPDM type 4. Targeted methylation sequencing revealed hypermethylation at the RILPL1 locus in unaffected individuals with ultralong expansion. Analysis of muscle samples showed no significant differences in RILPL1 mRNA or RILPL1 protein levels between patients and controls. Public CAGE-seq data indicated that alternative transcription start sites exist upstream of the RefSeq-annotated RILPL1 transcription start site. Strand-specific RNA-seq data revealed bidirectional transcription from the RILPL1 locus. Finally, fluorescence in situ hybridization/immunofluorescence staining showed that both sense and antisense transcripts formed RNA foci, and were co-localized with hnRNPA2B1 and p62 in the intranuclear inclusions of OPDM type 4 patients. INTERPRETATION: Our findings implicate abnormal GGC repeat expansions in the promoter region of RILPL1 as a novel genetic cause for OPDM, and suggest a methylation mechanism and a potential RNA toxicity mechanism are involved in OPDM type 4 pathogenesis. ANN NEUROL 2022;92:512-526.
Subject(s)
Muscular Dystrophies , Adult , Humans , In Situ Hybridization, Fluorescence , Intranuclear Inclusion Bodies/pathology , Muscular Dystrophies/genetics , Pedigree , RNA , Trinucleotide Repeat Expansion/geneticsSubject(s)
Muscular Diseases , Ubiquitin , Autophagy , Genotype , Humans , Muscle, Skeletal , Muscular Diseases/genetics , Perilipin-4/genetics , Phenotype , Ubiquitin/genetics , VacuolesABSTRACT
Background and Purpose: Hemoglobin is one of the main proteins in erythrocytes. There are significant correlations between low hemoglobin and white matter hyperintensities (WMH) and cognitive impairment. This study explored whether erythrocytopenia has predictive value for vascular cognitive impairment (VCI) in patients with WMH. Method: We conducted a cross-sectional study of 302 patients, including 62 with cerebral small vessel disease and 240 with stroke. Basic demographic data and fasting blood were collected. First, all patients were divided into normal cognition (NC), mild VCI (mVCI), and severe VCI (sVCI) groups (subgroups later) based on cognitive behavior scores. Second, all patients were divided into mild WMH (mWMH) and severe WMH (sWMH) groups based on Fazekas scores. The differences in blood markers between different groups or subgroups with different cognitive levels were analyzed by univariate analysis. Then, binary logistic regression was used to analyze the diagnostic value of erythrocyte counts for VCI in the sWMH group, and ordinal logistic regression was used to analyze the predictive value of multiple variables for different cognitive levels. Results: Univariate analysis showed that erythrocytes, hemoglobin, high-sensitivity C-reactive protein, retinol binding protein and prealbumin were potential blood markers for different cognitive levels in sWMH patients. Among them, erythrocytopenia has good predictive value for the diagnosis of mVCI (AUC = 0.685, P = 0.008) or sVCI (AUC = 0.699, P = 0.003) in patients with sWMH. Multivariate joint analysis showed that erythrocytes were an independent protective factor reducing the occurrence of VCI in patients with sWMH (OR = 0.633, P = 0.045). Even after adjusting for age, there was still a significant difference (P = 0.047). Conclusion: Erythrocytes are an independent protective factor for VCI in patients with sWMH. Promoting hematopoietic function may have potential value for prevention of cognitive decline in patients with cerebrovascular disease.
Subject(s)
Autoimmune Diseases of the Nervous System , Calcinosis , Nervous System Malformations , Autoimmune Diseases of the Nervous System/complications , Autoimmune Diseases of the Nervous System/diagnostic imaging , Autoimmune Diseases of the Nervous System/genetics , Calcinosis/complications , Calcinosis/diagnostic imaging , Calcinosis/genetics , Humans , Nervous System Malformations/complications , Nervous System Malformations/diagnostic imaging , Nervous System Malformations/geneticsABSTRACT
INTRODUCTION: Hereditary spastic paraplegias (HSPs) are uncommon but not rare neurodegenerative diseases. More than 100 pathogenic genes and loci related to spastic paraplegia symptoms have been reported. HSPs have the same core clinical features, including progressive spasticity in the lower limbs, though HSPs are heterogeneous (eg, clinical signs, MRI features, gene mutation). The age of onset varies greatly, from infant to adulthood. In addition, the slow and variable rates of disease progression in patients with HSP represent a substantial challenge for informative assessment of therapeutic efficacy. To address this, we are undertaking a prospective cohort study to investigate genetic-clinical characteristics, find surrogates for monitoring disease progress and identify clinical readouts for treatment. METHODS AND ANALYSIS: In this case-control cohort study, we will enrol 200 patients with HSP and 200 healthy individuals in parallel. Participants will be continuously assessed for 3 years at 12-month intervals. Six aspects, including clinical signs, genetic spectrum, cognitive competence, MRI features, potential biochemical indicators and nerve electrophysiological factors, will be assessed in detail. This study will observe clinical manifestations and disease severity based on different molecular mechanisms, including oxidative stress, cholesterol metabolism and microtubule dynamics, all of which have been proposed as potential treatment targets or modalities. The analysis will also assess disease progression in different types of HSPs and cellular pathways with a longitudinal study using t tests and χ2 tests. ETHICS AND DISSEMINATION: The study was granted ethics committee approval by the first affiliated hospital of Fujian Medical University (MRCTA, ECFAH of FMU (2019)194) in 2019. Findings will be disseminated via presentations and peer-reviewed publications. Dissemination will target different audiences, including national stakeholders, researchers from different disciplines and the general public. TRIAL REGISTRATION NUMBER: NCT04006418.
Subject(s)
Spastic Paraplegia, Hereditary , Adult , Case-Control Studies , China , Cohort Studies , Hospitals , Humans , Longitudinal Studies , Mutation , Prospective Studies , Spastic Paraplegia, Hereditary/diagnosis , Spastic Paraplegia, Hereditary/genetics , Spastic Paraplegia, Hereditary/pathologyABSTRACT
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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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.